自己翻译的罗氏tunel检测细胞凋亡试剂盒说明书
TUNEL细胞凋亡试剂盒内容及操作步骤
TUNEL细胞凋亡试剂盒内容及操作步骤--------------------------------------------------------------------------------凋亡细胞的原位末断转移酶标记法（TUNEL法）细胞凋亡的多步骤机制作用的最终环节是；细胞内源性核酸内切酶的激活而导致核染色质DNA双链的断裂。
大量DNA片段暴露出的3 羟基在末断转移酶(terminal deoxynucleotidyl transferase, TdT)或DNA多聚酶的作用下，与生物素或地高辛标记的核苷酸结合，最终借助与卵白素或抗地高辛抗体结合的荧光素或HRP，使凋亡细胞被特异性地标记和显示出来。
TUNEL细胞凋亡试剂盒由美国罗氏公司提供试剂1：酶浓缩溶液（Enzyme Solution）试剂2：标记溶液（Lable Solution）试剂3：转化剂-POD（Converter-POD）酶标记抗荧光素抗体（即用型）试验所需其它试剂：非石蜡切片:·冲洗缓冲液:磷酸盐缓冲液（PBS）·阻断溶液:0.3%H2O2甲醇溶液·固定溶液:4%多聚甲醛（溶剂pH7.4新鲜配制的PBS溶液）·渗透液:0.1%Triton甔-100（溶于新鲜配制的0.1%枸橼酸钠溶液）Triton X-100(聚乙二醇辛基苯基醚)名：曲拉通X-100，乳化剂OP分子式：C34H62O11石蜡切片:·二甲苯和乙醇（100%、95%、90%、80%、70%用蒸馏水稀释）·冲洗缓冲液:磷酸盐缓冲液（PBS）·蛋白酶K 工作液10~20mg/ml溶于10mM Tris/HCl（pH7.4~7.8）根据需要选择:·渗透液:0.1%TritonX-100（溶于新鲜配制的0.1%枸橼酸钠溶液）·胃酶溶液（0.25%-0.5%溶于HCl ，pH2）或胰酶·0.1M枸橼酸缓冲液，pH6，微波修复材料：微波炉，微波输出功率850W-2000W2 .选用中性甲醇固定的活检及实验动物标本，常规脱水，二甲苯透明，石蜡包埋。
TUNEL细胞凋亡检测试剂盒(显色法)说明书
TUNEL 细胞凋亡检测试剂盒(显色法)产品编号 产品名称包装 C1098TUNEL 细胞凋亡检测试剂盒(显色法)50次产品简介：碧云天生产的显色法TUNEL 细胞凋亡检测试剂盒(Colorimetric TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法。
对于待检测的细胞或组织样品，经过生物素标记和后续的DAB 显色等步骤，即可在普通光学显微镜下观察到凋亡细胞。
细胞在发生凋亡时，会激活一些DNA 内切酶，这些内切酶会切断核小体间的基因组DNA 。
细胞凋亡时抽提DNA 进行电泳检测，可以发现180-200bp 的DNA ladder 。
基因组DNA 断裂时，暴露的3'-OH 可以在末端脱氧核苷酸转移酶(Terminal Deoxynucleotidyl Transferase, TdT)的催化下加上生物素(Biotin)标记的dUTP(Biotin-dUTP)，随后和辣根过氧化物酶(HRP)标记的Streptavidin (Streptavidin-HRP)结合，最后在HRP 的催化下通过DAB 显色来显示凋亡细胞，从而可以通过普通光学显微镜检测到凋亡的细胞，这就是TUNEL(TdT-mediated dUTP Nick-End Labeling)法检测细胞凋亡的原理。
本试剂盒有如下优点。
(1) 高灵敏度：背景染色极低，阳性染色强，可以在单细胞水平检测到细胞凋亡，同时由于凋亡早期就有DNA 断裂，可以检测到早期的细胞凋亡。
(2) 特异性好：TUNEL 检测时通常更容易标记凋亡细胞，而不容易标记坏死细胞。
(3) 快速：仅需约2-3个小时即可完成。
(4) 应用范围广：可以用于检测冷冻或石蜡切片中的细胞凋亡情况，也可以检测培养的贴壁细胞或悬浮细胞的凋亡情况。
(5) 实测效果好：参考图1。
图1. 本试剂盒的检测效果图。
A. HeLa 细胞未处理或用DNase I 室温孵育10分钟后的检测效果图。
自己翻译的罗氏tunel检测细胞凋亡试剂盒说明书
罗氏tunel检测细胞凋亡试剂盒说明书注意：Label溶液含有甲次砷酸盐和二氯化钴，严禁吸入和食入。
反应悬浮物收集于密闭、不易碎、有明确标识的容器中，按有毒废物处理。
除上表所列试剂外，还需准备以下溶液。
下表列出每步所需物品概览：特异性：TUNEL反应优先标记凋亡产生的DNA链断裂，从而辨别凋亡与坏死、以及由抑制细胞生长的药物或放射线产生的primary DNA链断裂实验干扰：假阴性：在某些型式的凋亡细胞中DNA链断裂可能缺失或不完全。
空间位阻，如细胞外元件可能阻止TdT到达DNA断裂处。
两种情况均能产生假阴性。
假阳性：在坏死晚期，可能产生大量的DNA片段DNA链断裂也可能在具有高增殖和代谢活动的细胞中出现。
两种情况均能产生假阳性。
为确认细胞死亡的凋亡型式，应认真进行每种细胞的形态学检查凋亡过程中产生的形态学改变尤其特征形式，因此，对于可以结果进行解释时，细胞形态评估是一项重要的参数样本：细胞离心涂片和细胞涂片在chamber slides上培养的黏附细胞冰冻或福尔马林固定、石蜡包埋样本分析时间：2-3小时，除外培养、固定和渗透检测次数：一个试剂盒50T试剂盒存储/稳定性：未开封试剂盒储存于-15～-25℃可稳定至标签上标明的效期。
1 流程图：2 样品准备2.1 黏附细胞、细胞涂片和细胞离心涂片需准备的其他试剂：Washing buffer：磷酸盐缓冲液（PBS）Blocking buffer封闭溶液：甲醇稀释的3% H2O2Fixation solution固定溶液：PBS配制的4%多聚甲醛，ph 7.4，新鲜配制Permeabilisation solution 渗透液：0.1%Triton1）X-100溶于0.1%柠檬酸钠溶液中，新鲜配制步骤：下表描述了细胞固定、内源性过氧化物酶封闭和细胞渗透过程。
2.2 组织部分2.2.1 福尔马林-包埋组织福尔马林包埋组织的预处理：可按4种不同的方式预处理。
翻译好的罗氏公司Tunel试剂盒操作说明书
罗氏 (Roche)公司 Tunel 试剂盒操作说明书(In situ cell death detection kit-POD法)一、原理：TUNEL （TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA 的断裂情况。
其原理是荧光素（ fluorescein）标记的 dUTP 在脱氧核糖核苷酸末端转移酶（ TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂 DNA 的3’-OH 末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与 HRP 底物二氨基联苯胺（DAB ）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有 DNA 断裂，因而没有 3‘-OH 形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1 号（蓝盖） Enzyme Solution 酶溶液： TdT 10×、2号（紫盖） Label Solution 标记液：荧光素标记的 dUTP 1×、3号（棕瓶） Converter-POD:标记荧光素抗体的 HRP；自备试剂： PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB 工作液（临用前配制， 5 μl 20 ×DAB+1 μL 30%H2O2+94 μl PBS）、Proteinase K工作液（ 10-20 μg/ml in 10 mM Tris/HCl ，pH 7.4-8）或细胞通透液（0.1% Triton X-100 溶于 0.1% 柠檬酸钠，临用前配制）、苏木素或甲基绿、 DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl ，pH 7.5， 10 mM MgCl2 ，1 mg/ml BSA ）等。
翻译好的 罗氏公司Tunel试剂盒操作说明书
罗氏(Roche)公司Tunel试剂盒操作说明书(In situ cell death detection kit-POD法)一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）Enzyme Solution 酶溶液：TdT 10×、2号（紫盖）Label Solution标记液：荧光素标记的dUTP 1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5 μl 20×DAB+1μL 30%H2O2+94 μl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl，pH 7.4-8）或细胞通透液（0.1% Triton X-100 溶于0.1% 柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml–3 U/ml in 50 mM Tris-HCl，pH 7.5，10 mM MgCl2，1 mg/ml BSA）等。
罗氏 荧光 tunel (11684795910)说明书
In Situ Cell Death Detection Kit, Fluorescein
y Version 17
Content version: November 2012
Preface .....................................................................................................................................................2 Table of contents ................................................................................................................................................................. 2 Kit contents ............................................................................................................................................................................ 3 Introduction ............................................................................................................................................5 Product overview .................................................................................................................................................................. 5 Background information ................................................................................................................................................... 8 Procedures and required materials .................................................................................................10 Flow chart .............................................................................................................................................................................10 Preparation of sample material ....................................................................................................................................11 Cell suspension ..................................................................................................................................................................11 Adherent cells, cell smears, and cytospin preparations .....................................................................................12 Tissue sections ....................................................................................................................................................................12 Treatment of paraffin-embedded tissue ...................................................................................................................12 Treatment of cryopreserved tissue ..............................................................................................................................14 Labeling protocol ...............................................................................................................................................................15 Before you begin ................................................................................................................................................................15 Labeling protocol for cell suspensions ......................................................................................................................16 Labeling protocol for adherent cells, cell smears, cytospin preparations and tissues ...........................................................................................................................................................................17 Labeling protocol for difficult tissue ...........................................................................................................................18 Typical results ......................................................................................................................................19 Appendix ................................................................................................................................................20 Troubleshooting .................................................................................................................................................................20 References ............................................................................................................................................................................23 Related products ................................................................................................................................................................24
TUNEL细胞凋亡检测试剂盒罗氏
TUNEL细胞凋亡检测试剂盒罗⽒For research purposes only. Not for use for in vitro diagnosticprocedures for clinical diagnosis.In Situ Cell Death Detection Kit, PODKit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.Cat. No. 1 684 817Store at ?15 to ?25°C 1 Kit (50 tests)Instruction ManualVersion 3, January 20031. Preface1.1Table of contentsP reface(2)1.1.1Table of contents (2)(3)1.2 Kitcontents(5)2. Introduction2.1Product overview (5)(8)2.2 Backgroundinformation3. Procedures and required materials (10)3.1Flow chart (10)3.2Preparation of sample material (11)3.2.1Adherent cells, cell smears and cytospin preparations (11)(12)sections3.2.2 Tissue3.2.2.1 Treatment of paraffin-embedded tissue (12)3.2.2.2Treatment of cryopreserved tissue (14)3.3Labeling protocol (15)3.3.1 Before you begin (15)3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations, and tissues (16)3.3.3 Labeling protocol for difficult tissue (17)(18)conversion3.4 Signal(19)4. Appendix(19)4.1 Trouble-shooting(22)4.2 References(23)4.3 Relatedproducts1.2 KitcontentsCaution The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.Avoid exposure and obtain special instructions before use.When using do not eat, drink or smoke. After contact with skin, wash immediately with plenty of water. In case of accident or if you feelunwell seek medical advice immediately (show label where possible). Collect the supernatants from the labeling reactions in a tightly closed,non-breakable container and indicate contents. Discard as regulatedfor toxic waste.Kit contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution?Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer10× conc.5×50l2 violet Label Solution?Nucleotide mixture in reaction buffer1×conc.5 × 550 l3 yellow Converter-POD?Anti-fluorescein antibody, Fab frag-ment from sheep, conjugated withhorse-radish peroxidase (POD)Ready-to-use3.5mlAdditional equipment required In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.Detailed information is given in front of each procedure.Procedure Equipment Reagents Preparation of sample material (section 3.2)Adherent cells, cell smears and cytospinpreparations (section3.2.1)?Cryopreserved tissue (section 3.2.2.2)?Washing buffer: Phosphate buffered saline(PBS*)Blocking solution: 3% H2O2 in methanolFixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared ?Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.2.1)?Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)?Washing buffer: PBS*Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free0.1 M Citrate buffer, pH 6 for microwave irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)?Micrococcal nuclease or ?DNase I, grade I *Adherent cells, cell smears, cytospin preparations, and tissues (section 3.3.2) ?Parafilm orcoverslipsHumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.3)?Plastic jarMicrowaveHumidifiedchamberCitrate buffer, 0.1 M, pH 6.0.Washing buffer: PBS*Tris-HCl, 0.1 M pH 7.5, containing 3% BSA*and 20% normal bovine serumSignal conversion (section 3.4)Humidified chamber Parafilm or coverslip Washing buffer: PBS*DAB Metal Enhanced Substrate Set* or alternative POD substrates Mounting medium for light microscopy1.2 Kitcontents,continued2. Introduction2.1Product overviewTest principle Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Fig. 1: Test principleApplication The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:Detection of individual apoptotic cells in frozen and formalin fixedtissue sections in basic research and routine pathology.Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.Typing of cells undergoing cell death in heterogeneous populationsby double staining procedures (6).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in latestages of necrosis (4, 38).DNA strand breaks may also be prominent in cell populations withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained. To confirm apoptotic mode of cell death, themorphology of respective cells should be examined very carefully.Morphological changes during apoptosis have a characteristic pattern.Therefore evaluation of cell morphology is an important parameter insituations where there is any ambiguity regarding interpretation ofresults.Sample material?Cytospin and cell smear preparationsAdherent cells cultured on chamber slides (31)Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25,26, 29, 30, 32–34, 36, 39)Assay time2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections. Number of tests The kit is designed for 50 tests.Kit storage/ stability The unopened kit is stable at ?15 to ?25°C through the expiration date printed on the label. Reagent Storage and stabilityTUNEL reaction mixture The TUNEL reaction mixture should be pre-pared immediately before use and shouldnot be stored.Keep TUNEL reaction mixture on ice untiluse.Converter-POD Once thawed the Converter-POD solutionshould be stored at 2–8°C (maximum stabil-ity 6 months).Note: Do not freeze!Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at singlecell level at very early stages (1, 2, 6).Specific Preferential labeling of apoptosis versusnecrosis (3, 4).Fast Short assay time (2-3 h).Convenient?Reagents are provided in stable, opti-mized form.No dilution steps required.Flexible?Suitable for fixed cells and tissue. Thisallows accumulation, storage and trans-port of samples (2, 5).Double staining enables identification oftype and differentiation state of cellsundergoing apoptosis (6).Function-tested Every lot is function-tested on apoptoticcells in comparison to a master lot.2.2 BackgroundinformationCell death Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic pattern of structural changes in nucleus and cytoplasm,including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage tochromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant role in many pathological conditions like ischemia, stroke,heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).Identification of apoptosis Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4). Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).2.2 Backgroundinformation,continued3. Procedures and required materialsThe working procedure described below has been developed andpublished by R. Sgonc and colleagues (6). The main advantage of thissimple and rapid procedure is the use of fluorescein-dUTP to labelDNA strand breaks. This allows the detection of DNA fragmentationby fluorescence microscopy directly after the TUNEL reaction priorto the addition of the secondary anti-fluorescein-POD-conjugate.3.1Flow chartAssay procedure The assay procedure is explained in the following flow chart.Adherent cells, cell smears and cytospin preparations Cryopreservedtissue sectionsParaffin-embeddedtissue sections↓↓↓Fixation ?Dewaxation ?Rehydration ?ProteasetreatmentPermeabilisation of samples↓Addition of TUNEL reaction mixtureOPTIONAL: Analysis of samples by fluorescence microscopy↓Addition of Converter-PODAddition of Substrate solution↓Analysis of samples by light microscopy3.2Preparation of sample material3.2.1Adherent cells, cell smears and cytospin preparationsAdditional solutions required ?Washing buffer: Phosphate buffered saline (PBS)Blocking solution: 3% H2O2 in methanolFixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-paredPermeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)Procedure In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at 15-25°C.2Rinse slides with PBS.3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).6Proceed as described under 3.3.3.2.2 Tissue sections3.2.2.1 Treatment of paraffin-embedded tissuePretreatment of paraffin embedded tissue Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!The other 3 alternative procedures are also described in the following table (step 2).Additional solutions required ?Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water) Washing buffer: PBSProteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 g/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatmentsPermeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly preparedPepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free0.1 M Citrate buffer, pH 6 for the microwave irradiationProcedure In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g. by heating at 60°C followed by washing inxylene and rehydration through a graded series of ethanoland double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15-60 min at 37°C.3. Microwave irradiation ?Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH6.0.Apply 350 W microwave irradiation for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.2.1 Treatment of paraffin-embedded tissue, continued3.2.2.2Treatment of cryopreserved tissueAdditional solutions required ?Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-paredWashing buffer: PBSBlocking solution: 3% H2O2 in methanolPermeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue In the following table the pretreatment of cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at 15–25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at ?15 to ?25°C.3Incubate with Blocking solution for 10 min at 15–25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2–8°C).6Proceed as described under 3.3.3.3Labeling protocol 3.3.1 Before you beginPreparation of TUNEL reaction mixture One pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) issufficient for staining 10 samples by using 50 ?l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 ?l Label Solutionper control.Note : The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture onice until use.Additionalreagents required ?Micrococcal nuclease or ?DNase I, grade I (Cat. No. 104 132)Controls Two negative controls and a positive control should be included ineach experimental set up.StepAction 1Remove 100 ?l Label Solution (vial 2) for two negative con-trols.2Add total volume (50 ?l) of Enzyme solution (vial 1) to the remaining 450 ?l Label Solution in vial 2 to obtain 500 ?l TUNEL reaction mixture.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 ?l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control:Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM T ris-HCl, pH 7.5, 10 mM MgCl 2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations andtissuesAdditional equipment and solutions required ?Washing buffer: PBS ?Humidified chamber ?Parafilm or coverslip Procedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add50?l TUNEL reaction mixture on sample.Note: For the negative control add 50 ?l Label solution each.To ensure a homogeneous spread of TUNEL reaction mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.5Rinse slide 3 times with PBS.6Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelengthin the range of 450–500 nm and detection in the range of515–565 nm (green).3.3.3 Labeling protocol for difficult tissueAdditional equipment and solutions required ?Citrate buffer, 0.1 M, pH 6.0.Washing buffer: PBSTris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serumPlastic jarMicrowaveHumidified chamberProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3?Apply 750 W (high) microwave irradiation for 1 min.Cool rapidly by immediately adding 80 ml double dist.water (20–25°C).Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!4Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 MpH 7.5, containing 3% BSA and 20% normal bovineserum.5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.6Add50µl of TUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in thedark.8?Rinse slide(s) three times in PBS for 5 min each.Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in therange of 515–565 nm (green).3.4 SignalconversionAdditional equipment and solutions required ?Washing buffer: PBSHumidified chamberParafilm or coverslipDAB Substrate* (Cat. No. 1 718 096) or alternative POD substrate Mounting medium for light microscopy Procedure Please refer to the following table.Step Action1Dry area around sample.2Add50?l Converter-POD (vial 3) on sample.Note: To ensure a homogeneous spread of Converter-PODacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or cover slip during incuba-tion.3Incubate slide in a humidified chamber for 30 min at 37°C.4Rinse slide 3× with PBS.5Add 50–100 ?l DAB Substrate or alternative POD substrates.6Incubate slide for 10 min at 15–25°C.7Rinse slide 3× with PBS.8Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.Alternative: Samples can be counterstained prior to analysisby light microscope.4. Appendix4.1 Trouble-shootingThis table describes various troubleshooting parameters. Problem Step/Reagent of ProcedurePossible cause RecommendationNonspecific labeling Embedding of tissue UV-irradiation forpolymerization ofembedding material(e.g. methacrylate)leads to DNA strandbreaksTry different embedding materialor different polymerizationreagent.Fixation Acidic fixatives (e.g.methacarn, Carnoy’sfixative)Try 4% buffered paraformal-dehyde.Try formalin or glutaralde-hyde.TUNEL reaction TdT concentration toohighReduce concentration of TdT bydiluting it 1:2 up to 1:10 withTUNEL Dilution Buffer (Cat. No.1 966 06).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-PODBlock with normal anti-sheepserum.Block for 20 min with PBScontaining 3% BSA.Reduce concentration ofconverter solution to 50%. Nucleases Some tissues (e.g. smooth muscles)show DNA strandbreaks very soon aftertissue preparationFix tissue immediately afterorgan preparation.Perfuse fixative through livervein.Some enzymes arestill activeBlock with a solution containingddUTP and dATP.continued on next page。
翻译好的罗氏公司Tunel试剂盒操作说明方案
罗氏(R o c h e)公司T u n e l试剂盒操作说明书(Insitucelldeathdetectionkit-POD法)一、原理：TUNEL（TdT-mediateddUTPnickendlabeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdTEnzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radishperoxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）EnzymeSolution酶溶液：TdT10×、2号（紫盖）LabelSolution标记液：荧光素标记的dUTP1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5μl20×DAB+1μL30%H2O2+94μlPBS）、ProteinaseK工作液（10-20μg/mlin10mMTris/HCl，pH7.4-8）或细胞通透液（0.1%TritonX-100溶于0.1%柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase1（3000U/ml–3U/mlin50mMTris-HCl，pH7.5，10mMMgCl2，1mg/mlBSA）等。
罗氏公司TUNEL细胞凋亡检测程序 (In situ cell death detection kit-POD法)
Caution
Kit contents
Enzyme Solution • Terminal deoxynucleotidyl transferase from calf thymus (EC 2.7.7.31), recombinant in E. coli, in storage buffer • 10× conc. • 5 × 50 l Label Solution • Nucleotide mixture in reaction buffer • 1× conc. • 5 × 550 l • Anti-fluorescein antibody, Fab fragment from sheep, conjugated with horse-radish peroxidase (POD) • Ready-to-use • 3.5 ml
2 violet 3 yellow
Converterontents, continued
Additional equipment required Procedure
In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures. Detailed information is given in front of each procedure. Equipment Reagents • Washing buffer: Phosphate buffered saline (PBS*) • Blocking solution: 3% H2O2 in methanol • Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly prepared • Permeabilisation solution: 0.1% Triton X100 in 0.1% sodium citrate, freshly prepared (6) • Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water) • Washing buffer: PBS* • Proteinase K*, nuclease free, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments • Permeabilisation solution: (0.1% Triton1) X– 100, 0.1% sodium citrate) , freshly prepared • Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free • 0.1 M Citrate buffer, pH 6 for microwave irradiation • Micrococcal nuclease or • DNase I, recombinant, grade I * • Parafilm or Washing buffer: PBS* coverslips • Humidified chamber • Plastic jar • Citrate buffer, 0.1 M, pH 6.0. • Microwave • Washing buffer: PBS* • Humidified • Tris-HCl, 0.1 M pH 7.5, containing 3% BSA* chamber and 20% normal bovine serum • Humidified • Washing buffer: PBS* chamber • DAB Metal Enhanced Substrate Set* or • Parafilm or alternative POD substrates coverslip • Mounting medium for light microscopy
TUNEL说明书
TUNEL说明书1 介绍TUNEL是提供单细胞水平细胞凋亡的稳定系统，能够迅速、快捷、精确的检测出凋亡细胞。
该试剂盒可以通过测定核DNA片段检测组织切片和培养细胞的凋亡细胞。
多数高等真核生物的细胞都通过启动自身的细胞自杀程序实现程序性死亡或细胞凋亡。
凋亡在发育、内环境稳定和一些疾病中具有重要作用。
凋亡具有某些特定的形态学特征，包括细胞膜起泡，细胞核和细胞质固缩，染色质浓缩，并且不发生局部炎症反应。
细胞死亡与此相反，它的特点是细胞肿胀，染色质絮凝，细胞膜完整性破坏，细胞溶解和产生及局部炎症反应。
凋亡过程中，内源性Ca2+、Mg2+依赖性核酸内切酶被激活，DNA被降解而形成末端为3’－OH、含180～200碱基对的不同倍数的核苷酸片段。
TUNEL 可用于多种细胞凋亡的检测，已经经过验证的应用范围: Vibratome® 神经组织切片, Jurkat 细胞, HL-60细胞这本技术小册子包括检测组织切片和茴香霉素诱导的HL-60细胞的细胞凋亡。
检测原理: DeadEnd™ Colorimetric TUNEL 系统使用改良的TUNEL (TdT-mediated dUTP Nick-End Labeling)对凋亡细胞的断裂DNA进行末端标记。
使用末端脱氧核苷转移酶(TdT)将生物素标记的核苷被掺入到DNA的3′-OH末端。
然后，辣根过氧化物酶标记的链霉亲和素(Streptavidin HRP)结合在上述生物素标记的核苷上，可以通过过氧化物酶的底物——过氧化氢和稳定的显色剂氨基联苯胺(DAB)检测到。
用这种程序，凋亡细胞的核被染成深棕色。
2 产品内容G7361平衡缓冲液（G327B）——4.8ml末端脱氧核苷酰酶酸转移酶（M828B）——20ul抗生物素蛋白链菌素辣根过氧化物酶（G714A）——40ul生物素化的核苷混合物（G715A）——20ul蛋白酶K（V302A）——10mgG7362塑料盖玻片（G326B）——2020X SSC（G329B）——20mlDAB 20X 色原体（G716A）——200ul20X DAB底物缓冲液（G717A）——200ul20X过氧化氢（G718A）——200ul储存条件: 将平衡缓冲液, TdT酶, 生物素标记的核苷混合物和蛋白酶K 储存于–20°C。
TUNEL细胞凋亡检测试剂盒罗氏
TUNEL细胞凋亡检测试剂盒罗⽒For research purposes only. Not for use for in vitro diagnosticprocedures for clinical diagnosis.In Situ Cell Death Detection Kit, PODKit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.Cat. No. 1 684 817Store at ?15 to ?25°C 1 Kit (50 tests)Instruction ManualVersion 3, January 20031. Preface1.1Table of contentsP reface(2)1.1.1Table of contents (2)(3)1.2 Kitcontents(5)2. Introduction2.1Product overview (5)(8)2.2 Backgroundinformation3. Procedures and required materials (10)3.1Flow chart (10)3.2Preparation of sample material (11)3.2.1Adherent cells, cell smears and cytospin preparations (11)(12)sections3.2.2 Tissue3.2.2.1 Treatment of paraffin-embedded tissue (12)3.2.2.2Treatment of cryopreserved tissue (14)3.3Labeling protocol (15)3.3.1 Before you begin (15)3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations, and tissues (16)3.3.3 Labeling protocol for difficult tissue (17)(18)conversion3.4 Signal(19)4. Appendix(19)4.1 Trouble-shooting(22)4.2 References(23)4.3 Relatedproducts1.2 KitcontentsCaution The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.Avoid exposure and obtain special instructions before use.When using do not eat, drink or smoke. After contact with skin, wash immediately with plenty of water. In case of accident or if you feelunwell seek medical advice immediately (show label where possible). Collect the supernatants from the labeling reactions in a tightly closed,non-breakable container and indicate contents. Discard as regulatedfor toxic waste.Kit contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution?Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer10× conc.5×50l2 violet Label Solution?Nucleotide mixture in reaction buffer1×conc.5 × 550 l3 yellow Converter-POD?Anti-fluorescein antibody, Fab frag-ment from sheep, conjugated withhorse-radish peroxidase (POD)Ready-to-use3.5mlAdditional equipment required In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.Detailed information is given in front of each procedure.Procedure Equipment Reagents Preparation of sample material (section 3.2)Adherent cells, cell smears and cytospinpreparations (section3.2.1)?Cryopreserved tissue (section 3.2.2.2)?Washing buffer: Phosphate buffered saline(PBS*)Blocking solution: 3% H2O2 in methanolFixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared ?Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.2.1)?Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)?Washing buffer: PBS*Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free0.1 M Citrate buffer, pH 6 for microwave irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)?Micrococcal nuclease or ?DNase I, grade I *Adherent cells, cell smears, cytospin preparations, and tissues (section 3.3.2) ?Parafilm orcoverslipsHumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.3)?Plastic jarMicrowaveHumidifiedchamberCitrate buffer, 0.1 M, pH 6.0.Washing buffer: PBS*Tris-HCl, 0.1 M pH 7.5, containing 3% BSA*and 20% normal bovine serumSignal conversion (section 3.4)Humidified chamber Parafilm or coverslip Washing buffer: PBS*DAB Metal Enhanced Substrate Set* or alternative POD substrates Mounting medium for light microscopy1.2 Kitcontents,continued2. Introduction2.1Product overviewTest principle Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Fig. 1: Test principleApplication The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:Detection of individual apoptotic cells in frozen and formalin fixedtissue sections in basic research and routine pathology.Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.Typing of cells undergoing cell death in heterogeneous populationsby double staining procedures (6).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in latestages of necrosis (4, 38).DNA strand breaks may also be prominent in cell populations withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained. To confirm apoptotic mode of cell death, themorphology of respective cells should be examined very carefully.Morphological changes during apoptosis have a characteristic pattern.Therefore evaluation of cell morphology is an important parameter insituations where there is any ambiguity regarding interpretation ofresults.Sample material?Cytospin and cell smear preparationsAdherent cells cultured on chamber slides (31)Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25,26, 29, 30, 32–34, 36, 39)Assay time2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections. Number of tests The kit is designed for 50 tests.Kit storage/ stability The unopened kit is stable at ?15 to ?25°C through the expiration date printed on the label. Reagent Storage and stabilityTUNEL reaction mixture The TUNEL reaction mixture should be pre-pared immediately before use and shouldnot be stored.Keep TUNEL reaction mixture on ice untiluse.Converter-POD Once thawed the Converter-POD solutionshould be stored at 2–8°C (maximum stabil-ity 6 months).Note: Do not freeze!Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at singlecell level at very early stages (1, 2, 6).Specific Preferential labeling of apoptosis versusnecrosis (3, 4).Fast Short assay time (2-3 h).Convenient?Reagents are provided in stable, opti-mized form.No dilution steps required.Flexible?Suitable for fixed cells and tissue. Thisallows accumulation, storage and trans-port of samples (2, 5).Double staining enables identification oftype and differentiation state of cellsundergoing apoptosis (6).Function-tested Every lot is function-tested on apoptoticcells in comparison to a master lot.2.2 BackgroundinformationCell death Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic pattern of structural changes in nucleus and cytoplasm,including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage tochromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant role in many pathological conditions like ischemia, stroke,heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).Identification of apoptosis Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4). Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).2.2 Backgroundinformation,continued3. Procedures and required materialsThe working procedure described below has been developed andpublished by R. Sgonc and colleagues (6). The main advantage of thissimple and rapid procedure is the use of fluorescein-dUTP to labelDNA strand breaks. This allows the detection of DNA fragmentationby fluorescence microscopy directly after the TUNEL reaction priorto the addition of the secondary anti-fluorescein-POD-conjugate.3.1Flow chartAssay procedure The assay procedure is explained in the following flow chart.Adherent cells, cell smears and cytospin preparations Cryopreservedtissue sectionsParaffin-embeddedtissue sections↓↓↓Fixation ?Dewaxation ?Rehydration ?ProteasetreatmentPermeabilisation of samples↓Addition of TUNEL reaction mixtureOPTIONAL: Analysis of samples by fluorescence microscopy↓Addition of Converter-PODAddition of Substrate solution↓Analysis of samples by light microscopy3.2Preparation of sample material3.2.1Adherent cells, cell smears and cytospin preparationsAdditional solutions required ?Washing buffer: Phosphate buffered saline (PBS)Blocking solution: 3% H2O2 in methanolFixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-paredPermeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)Procedure In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at 15-25°C.2Rinse slides with PBS.3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).6Proceed as described under 3.3.3.2.2 Tissue sections3.2.2.1 Treatment of paraffin-embedded tissuePretreatment of paraffin embedded tissue Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!The other 3 alternative procedures are also described in the following table (step 2).Additional solutions required ?Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water) Washing buffer: PBSProteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 g/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatmentsPermeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly preparedPepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free0.1 M Citrate buffer, pH 6 for the microwave irradiationProcedure In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g. by heating at 60°C followed by washing inxylene and rehydration through a graded series of ethanoland double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15-60 min at 37°C.3. Microwave irradiation ?Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH6.0.Apply 350 W microwave irradiation for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.2.1 Treatment of paraffin-embedded tissue, continued3.2.2.2Treatment of cryopreserved tissueAdditional solutions required ?Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-paredWashing buffer: PBSBlocking solution: 3% H2O2 in methanolPermeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue In the following table the pretreatment of cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at 15–25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at ?15 to ?25°C.3Incubate with Blocking solution for 10 min at 15–25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2–8°C).6Proceed as described under 3.3.3.3Labeling protocol 3.3.1 Before you beginPreparation of TUNEL reaction mixture One pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) issufficient for staining 10 samples by using 50 ?l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 ?l Label Solutionper control.Note : The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture onice until use.Additionalreagents required ?Micrococcal nuclease or ?DNase I, grade I (Cat. No. 104 132)Controls Two negative controls and a positive control should be included ineach experimental set up.StepAction 1Remove 100 ?l Label Solution (vial 2) for two negative con-trols.2Add total volume (50 ?l) of Enzyme solution (vial 1) to the remaining 450 ?l Label Solution in vial 2 to obtain 500 ?l TUNEL reaction mixture.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 ?l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control:Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM T ris-HCl, pH 7.5, 10 mM MgCl 2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations andtissuesAdditional equipment and solutions required ?Washing buffer: PBS ?Humidified chamber ?Parafilm or coverslip Procedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add50?l TUNEL reaction mixture on sample.Note: For the negative control add 50 ?l Label solution each.To ensure a homogeneous spread of TUNEL reaction mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.5Rinse slide 3 times with PBS.6Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelengthin the range of 450–500 nm and detection in the range of515–565 nm (green).3.3.3 Labeling protocol for difficult tissueAdditional equipment and solutions required ?Citrate buffer, 0.1 M, pH 6.0.Washing buffer: PBSTris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serumPlastic jarMicrowaveHumidified chamberProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3?Apply 750 W (high) microwave irradiation for 1 min.Cool rapidly by immediately adding 80 ml double dist.water (20–25°C).Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!4Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 MpH 7.5, containing 3% BSA and 20% normal bovineserum.5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.6Add50µl of TUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in thedark.8?Rinse slide(s) three times in PBS for 5 min each.Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in therange of 515–565 nm (green).3.4 SignalconversionAdditional equipment and solutions required ?Washing buffer: PBSHumidified chamberParafilm or coverslipDAB Substrate* (Cat. No. 1 718 096) or alternative POD substrate Mounting medium for light microscopy Procedure Please refer to the following table.Step Action1Dry area around sample.2Add50?l Converter-POD (vial 3) on sample.Note: To ensure a homogeneous spread of Converter-PODacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or cover slip during incuba-tion.3Incubate slide in a humidified chamber for 30 min at 37°C.4Rinse slide 3× with PBS.5Add 50–100 ?l DAB Substrate or alternative POD substrates.6Incubate slide for 10 min at 15–25°C.7Rinse slide 3× with PBS.8Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.Alternative: Samples can be counterstained prior to analysisby light microscope.4. Appendix4.1 Trouble-shootingThis table describes various troubleshooting parameters. Problem Step/Reagent of ProcedurePossible cause RecommendationNonspecific labeling Embedding of tissue UV-irradiation forpolymerization ofembedding material(e.g. methacrylate)leads to DNA strandbreaksTry different embedding materialor different polymerizationreagent.Fixation Acidic fixatives (e.g.methacarn, Carnoy’sfixative)Try 4% buffered paraformal-dehyde.Try formalin or glutaralde-hyde.TUNEL reaction TdT concentration toohighReduce concentration of TdT bydiluting it 1:2 up to 1:10 withTUNEL Dilution Buffer (Cat. No.1 966 06).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-PODBlock with normal anti-sheepserum.Block for 20 min with PBScontaining 3% BSA.Reduce concentration ofconverter solution to 50%. Nucleases Some tissues (e.g. smooth muscles)show DNA strandbreaks very soon aftertissue preparationFix tissue immediately afterorgan preparation.Perfuse fixative through livervein.Some enzymes arestill activeBlock with a solution containingddUTP and dATP.continued on next page。
TUNEL细胞凋亡检测试剂盒 罗氏
For research purposes only. Not for use for in vitro diagnosticprocedures for clinical diagnosis.In Situ Cell Death Detection Kit, PODKit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.Cat. No. 1 684 817Store at Ϫ15 to Ϫ25°C 1 Kit (50 tests)Instruction ManualVersion 3, January 20031. Preface1.1Table of contentsP reface (2)1.1.1Table of contents (2) (3)1.2 Kitcontents (5)2. Introduction2.1Product overview (5) (8)2.2 Backgroundinformation3. Procedures and required materials (10)3.1Flow chart (10)3.2Preparation of sample material (11)3.2.1Adherent cells, cell smears and cytospin preparations (11) (12)sections3.2.2 Tissue3.2.2.1 Treatment of paraffin-embedded tissue (12)3.2.2.2Treatment of cryopreserved tissue (14)3.3Labeling protocol (15)3.3.1 Before you begin (15)3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations,and tissues (16)3.3.3 Labeling protocol for difficult tissue (17) (18)conversion3.4 Signal (19)4. Appendix (19)4.1 Trouble-shooting (22)4.2 References (23)4.3 Relatedproducts1.2 KitcontentsCaution The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.Avoid exposure and obtain special instructions before use.When using do not eat, drink or smoke. After contact with skin, washimmediately with plenty of water. In case of accident or if you feelunwell seek medical advice immediately (show label where possible).Collect the supernatants from the labeling reactions in a tightly closed,non-breakable container and indicate contents. Discard as regulatedfor toxic waste.Kit contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution•Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer•10× conc.•5×50l2 violet Label Solution•Nucleotide mixture in reaction buffer•1×conc.• 5 × 550 l3 yellow Converter-POD•Anti-fluorescein antibody, Fab frag-ment from sheep, conjugated withhorse-radish peroxidase (POD)•Ready-to-use• 3.5mlAdditional equipment required In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.Detailed information is given in front of each procedure.Procedure Equipment Reagents Preparation of sample material (section 3.2)•Adherent cells, cell smears and cytospinpreparations (section3.2.1)•Cryopreserved tissue (section 3.2.2.2)•Washing buffer: Phosphate buffered saline(PBS*)•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared •Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.2.1)•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)•Washing buffer: PBS*•Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments•Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared •Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free•0.1 M Citrate buffer, pH 6 for microwave irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)•Micrococcal nuclease or •DNase I, grade I *Adherent cells, cell smears, cytospin preparations, and tissues (section 3.3.2) •Parafilm orcoverslips•HumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.3)•Plastic jar•Microwave•Humidifiedchamber•Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS*•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA*and 20% normal bovine serumSignal conversion (section 3.4)•Humidified chamber •Parafilm or coverslip •Washing buffer: PBS*•DAB Metal Enhanced Substrate Set* or alternative POD substrates •Mounting medium for light microscopy1.2 Kitcontents,continued2. Introduction2.1Product overviewTest principle Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Fig. 1: Test principleApplication The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:•Detection of individual apoptotic cells in frozen and formalin fixedtissue sections in basic research and routine pathology.•Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.•Typing of cells undergoing cell death in heterogeneous populationsby double staining procedures (6).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in latestages of necrosis (4, 38).DNA strand breaks may also be prominent in cell populations withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained. To confirm apoptotic mode of cell death, themorphology of respective cells should be examined very carefully.Morphological changes during apoptosis have a characteristic pattern.Therefore evaluation of cell morphology is an important parameter insituations where there is any ambiguity regarding interpretation ofresults.Sample material•Cytospin and cell smear preparations•Adherent cells cultured on chamber slides (31)•Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25,26, 29, 30, 32–34, 36, 39)Assay time2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections.Number of tests The kit is designed for 50 tests.Kit storage/ stability The unopened kit is stable at Ϫ15 to Ϫ25°C through the expiration date printed on the label.Reagent Storage and stabilityTUNEL reaction mixture The TUNEL reaction mixture should be pre-pared immediately before use and shouldnot be stored.Keep TUNEL reaction mixture on ice untiluse.Converter-POD Once thawed the Converter-POD solutionshould be stored at 2–8°C (maximum stabil-ity 6 months).Note: Do not freeze!Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at singlecell level at very early stages (1, 2, 6).Specific Preferential labeling of apoptosis versusnecrosis (3, 4).Fast Short assay time (2-3 h).Convenient•Reagents are provided in stable, opti-mized form.•No dilution steps required.Flexible•Suitable for fixed cells and tissue. Thisallows accumulation, storage and trans-port of samples (2, 5).•Double staining enables identification oftype and differentiation state of cellsundergoing apoptosis (6).Function-tested Every lot is function-tested on apoptoticcells in comparison to a master lot.2.2 BackgroundinformationCell death Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic pattern of structural changes in nucleus and cytoplasm,including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage tochromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant role in many pathological conditions like ischemia, stroke,heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).Identification of apoptosis Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:•Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4).•Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).•TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).2.2 Backgroundinformation,continued3. Procedures and required materialsThe working procedure described below has been developed andpublished by R. Sgonc and colleagues (6). The main advantage of thissimple and rapid procedure is the use of fluorescein-dUTP to labelDNA strand breaks. This allows the detection of DNA fragmentationby fluorescence microscopy directly after the TUNEL reaction priorto the addition of the secondary anti-fluorescein-POD-conjugate.3.1Flow chartAssay procedure The assay procedure is explained in the following flow chart.Adherent cells, cell smears and cytospin preparations Cryopreservedtissue sectionsParaffin-embeddedtissue sections↓↓↓Fixation •Dewaxation •Rehydration •ProteasetreatmentPermeabilisation of samples↓Addition of TUNEL reaction mixtureOPTIONAL: Analysis of samples by fluorescence microscopy↓Addition of Converter-PODAddition of Substrate solution↓Analysis of samples by light microscopy3.2Preparation of sample material3.2.1Adherent cells, cell smears and cytospin preparationsAdditional solutions required •Washing buffer: Phosphate buffered saline (PBS)•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared•Permeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)Procedure In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at 15-25°C.2Rinse slides with PBS.3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).6Proceed as described under 3.3.3.2.2 Tissue sections3.2.2.1 Treatment of paraffin-embedded tissuePretreatment of paraffin embedded tissue Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!The other 3 alternative procedures are also described in the following table (step 2).Additional solutions required •Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water)•Washing buffer: PBS•Proteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 g/ml in 10 mM Tris/HCl, pH 7.4-8]Alternative treatments•Permeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly prepared•Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free•0.1 M Citrate buffer, pH 6 for the microwave irradiationProcedure In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g. by heating at 60°C followed by washing inxylene and rehydration through a graded series of ethanoland double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15-60 min at 37°C.3. Microwave irradiation •Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH6.0.•Apply 350 W microwave irradiation for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.2.1 Treatment of paraffin-embedded tissue, continued3.2.2.2Treatment of cryopreserved tissueAdditional solutions required •Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared•Washing buffer: PBS•Blocking solution: 3% H2O2 in methanol•Permeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue In the following table the pretreatment of cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at 15–25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at Ϫ15 to Ϫ25°C.3Incubate with Blocking solution for 10 min at 15–25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2–8°C).6Proceed as described under 3.3.3.3Labeling protocol 3.3.1Before you beginPreparation of TUNEL reaction mixtureOne pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) is sufficient for staining 10 samples by using 50 l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 l Label Solution per control.Note : The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.Additionalreagents required •Micrococcal nuclease or •DNase I, grade I (Cat. No. 104 132)ControlsTwo negative controls and a positive control should be included in each experimental set up.Step Action1Remove 100 l Label Solution (vial 2) for two negative con-trols.2Add total volume (50 l) of Enzyme solution (vial 1) to the remaining 450 l Label Solution in vial 2 to obtain 500 l TUNEL reaction mixture.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control:Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM T ris-HCl, pH 7.5, 10 mM MgCl 2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations andtissuesAdditional equipment and solutions required •Washing buffer: PBS •Humidified chamber •Parafilm or coverslipProcedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add50l TUNEL reaction mixture on sample.Note: For the negative control add 50 l Label solution each.To ensure a homogeneous spread of TUNEL reaction mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.5Rinse slide 3 times with PBS.6Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelengthin the range of 450–500 nm and detection in the range of515–565 nm (green).3.3.3 Labeling protocol for difficult tissueAdditional equipment and solutions required •Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum•Plastic jar•Microwave•Humidified chamberProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3•Apply 750 W (high) microwave irradiation for 1 min.•Cool rapidly by immediately adding 80 ml double dist.water (20–25°C).•Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!4Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 MpH 7.5, containing 3% BSA and 20% normal bovineserum.5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.6Add50µl of TUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in thedark.8•Rinse slide(s) three times in PBS for 5 min each.•Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in therange of 515–565 nm (green).3.4 SignalconversionAdditional equipment and solutions required •Washing buffer: PBS•Humidified chamber•Parafilm or coverslip•DAB Substrate* (Cat. No. 1 718 096) or alternative POD substrate •Mounting medium for light microscopyProcedure Please refer to the following table.Step Action1Dry area around sample.2Add50l Converter-POD (vial 3) on sample.Note: To ensure a homogeneous spread of Converter-PODacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or cover slip during incuba-tion.3Incubate slide in a humidified chamber for 30 min at 37°C.4Rinse slide 3× with PBS.5Add 50–100 l DAB Substrate or alternative POD substrates.6Incubate slide for 10 min at 15–25°C.7Rinse slide 3× with PBS.8Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.Alternative: Samples can be counterstained prior to analysisby light microscope.4. Appendix4.1 Trouble-shootingThis table describes various troubleshooting parameters. Problem Step/Reagent ofProcedurePossible cause RecommendationNonspecific labeling Embedding of tissue UV-irradiation forpolymerization ofembedding material(e.g. methacrylate)leads to DNA strandbreaksTry different embedding materialor different polymerizationreagent.Fixation Acidic fixatives (e.g.methacarn, Carnoy’sfixative)•Try 4% buffered paraformal-dehyde.•Try formalin or glutaralde-hyde.TUNEL reaction TdT concentration toohighReduce concentration of TdT bydiluting it 1:2 up to 1:10 withTUNEL Dilution Buffer (Cat. No.1 966 06).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-POD•Block with normal anti-sheepserum.•Block for 20 min with PBScontaining 3% BSA.•Reduce concentration ofconverter solution to 50%. Nucleases Some tissues (e.g.smooth muscles)show DNA strandbreaks very soon aftertissue preparation•Fix tissue immediately afterorgan preparation.•Perfuse fixative through livervein.Some enzymes arestill activeBlock with a solution containingddUTP and dATP.continued on next pageHigh back-ground Fixation Formalin fixation leadsto a yellowish stainingof cells containingmelanin precursorsTry methanol for fixation buttake into account that this mightlead to reduced sensitivity.TUNEL reaction Concentration oflabeling mix is toohigh for mamma car-cinomaReduce concentration of label-ing mix to 50% by diluting withTUNEL Dilution Buffer (Cat. No.1 966 006).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-POD•Block with normal anti-sheepserum.•Block for 20 min with PBScontaining 3% BSA.•Reduce concentration ofconverter solution to 50%. Sample Mycoplasma contami-nationMycoplasma detection Kit (Cat.No. 1 296 7449).Highly proliferatingcellsDouble staining e.g. withAnnexin-V-Fluos (Cat. No. 1 828681).Note: Measuring via microplatereader not possible because oftoo high background.Low labeling Fixation Ethanol and methanolcan lead to low label-ing (nucleosomes arenot cross-linked withproteins during fixa-tion and are lost dur-ing the proceduresteps)•Try 4% buffered paraformal-dehyde.•Try formalin or glutaralde-hyde.Extensive fixationleads to excessivecrosslinking of pro-teins•Reduce fixation time.•Try 2% buffered paraformal-dehyde.Permeabilisation Permeabilisation tooshort so that reagentscan’t reach their tar-get molecules•Increase incubation time.•Incubate at higher tempera-ture (e.g. 15–25°C).•Try Proteinase K (concentra-tion and time has to be opti-mized for each type oftissue).•Try 0.1 M sodium citrate at70°C for 30 min.continued on next pageProblem Step/Reagent ofProcedure Possible cause Recommendation4.1Trouble-shooting, continuedParaffin-embedding Accessibility forreagents is too low •Treat tissue sections afterdewaxing with Proteinase K (concentration, time andtemperature have to be opti-mized for each type of tis-sue).•Try microwave irradiation at370 W (low) for 5 min in200ml 0.1 M Citrate bufferpH 6.0 (has to be optimizedfor each type of tissue).No signal on positive control DNase treatment Concentration ofDNase is too low•For cryosections apply 3 U/mlDNase I, grade I.•For paraffin-embedded tissuesections apply 1500 U/mlDNase I, grade I.•In general, use 1 U/mlDNase I, grade I, dissolved in10 mM Tris-HCl pH 7.4 con-taining 10 mM NaCl, 5 mMMnCl2, 0.1 mM CaCl2, 25 mMKCl and incubate 30 min at37°C.•Alternative buffer 50 mMTris- HCl pH 7.5 containing1mM MgCl2 and 1 mg/mlBSA.Weak sig-nals Counterstaining Not suitable dye•Counterstaining with 5%methyl green in 0,1 M veronalacetate, pH 4.0 or Hematoxi-lin is possible (43).•Double-staining with propid-ium iodide is possible butonly for detection of morpho-logical cell changes.Problem Step/Reagent ofProcedure Possible cause Recommendation4.1Trouble-shooting, continued4.2 References1Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. (1992) J. Cell Biol. 119, 493–501.2Gorczyca, W., Gong, J. & Darzynkiewicz, Z. (1993) Cancer Res. 53, 1945–1951.3Gorczyca, W. et al. (1993) Leukemi a 7, 659–670.4Gold, R. et al. (1994) Lab. Invest. 71, 219.5Gorczyca, W. et al. (1994) Cytometry 15, 169–175.6Sgonc, R. et al. (1994) Trends Genetics 10, 41–42.7Schmied, M. et al. (1993) Am. J. Pathol. 143, 446–452.8Wyllie, A. H. et al. (1980) Int. Rev. Cytol. 68, 251.9Kerr, J. F. R. et al. (1972) Br. J. Cancer 26, 239–257.10Duvall, E. & Wyllie, A. H. (1986) Immunol. To day 7, 115.11Compton, M. M. (1992) Canc. Metastasis Rev. 11, 105–119.12Allen, P. D., Bustin, S. A. & Newland, A. C. (1993) Blood Reviews 7, 63–73.13Cohen, J. J. & Duke, R. C. (1992) Annu. Rev. Immunol. 10, 267–293.14Clarke, P. G. H. (1990) Anat. Embryol. 181, 195–213.15Johnson, E. M. & Deckwerth, T. L. (1993) Annu. Rev. Neurosci. 16, 31–46.16Batistatou, A. & Greene, L. A. (1993) J. Cell Biol. 122, 523–532.17Strange, R. et al. (1992) Development 115, 49–58.18Carson, D. A. & Ribeiro, J. M. (1993) Lancet 341, 1251–1254.19Edgington, S. M. (1993) Biotechnology 11, 787–792.20Gougeon. M.-L. & Montagnier, L. (1993) Science 260, 1269–1270.21Hickman, J. A. (1992) Cancer Metastasis Rev. 11, 121–139.22Afanasyev, V. N. et al. (1993) Cytometry 14, 603–609.23Bryson, G. J., Harmon, B. V. & Collins, R. J. (1994) Immunology Cell Biology 72,35–4124Darzynkiewicz, Z. et al. (1992) Cytometry 13, 795–808.25Ando, K. et al. (1994) J. Immunol. 152, 3245–3253.26Berges, R. R. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8910– 8914.27Gorczyca, W. et al. (1992) Int. J. Oncol. 1, 639–648.28Gorczyca, W. et al. (1993) Exp. Cell Res. 207, 202–205.29Billig, H., Furuta, I. & Hsueh, A. J. W. (1994) Endocrinology 134, 245–252.30MacManus, J. P. et al. (1993) Neurosci. Lett. 164, 89–92.31Mochizuki, H. et al. (1994) Neurosci. Lett. 170, 191–194.32Oberhammer, F. et al. (1993) Hepatology 18, 1238–1246.33Portera-Cailliau, C. (1994) Proc. Natl. Acad. Sci. USA 91, 974 –978.34Preston, G. A. et al. (1994) Cancer Res. 54, 4214–4223.35Weller, M. et al. (1994) Eur. J. Immunol. 24, 1293–1300.36Zager, R.A. et al. (1994) J. Am. Soc. Nephrol. 4, 1588–1597.37Cohen, G. M. et al. (1992) Biochem. J. 286, 331–334.38Collins, R. J. et al. (1992) Int. J. Rad. Biol. 61, 451–453.39Sei, Y. et al. (1994) Neurosci. Lett. 171, 179–182.40Ansari, B. et al. (1993) J. Pathol. 170, 1–8.41Gold, R. et al. (1993) J. Histochem. Cytochem. 41, 1023–1030.42Negoescu, A. et.al. (1998) Biochemica3, 34-41.43Umermura, S. et al. (1996) J. Histochem. Cytochem. 44, 125-132 .。
TUNEL细胞凋亡检测试剂盒 罗氏
For research purposes only. Not for use for in vitro diagnosticprocedures for clinical diagnosis.In Situ Cell Death Detection Kit, PODKit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.Cat. No. 1 684 817Store at Ϫ15 to Ϫ25°C 1 Kit (50 tests)Instruction ManualVersion 3, January 20031. Preface1.1Table of contentsP reface (2)1.1.1Table of contents (2) (3)1.2 Kitcontents (5)2. Introduction2.1Product overview (5) (8)2.2 Backgroundinformation3. Procedures and required materials (10)3.1Flow chart (10)3.2Preparation of sample material (11)3.2.1Adherent cells, cell smears and cytospin preparations (11) (12)sections3.2.2 Tissue3.2.2.1 Treatment of paraffin-embedded tissue (12)3.2.2.2Treatment of cryopreserved tissue (14)3.3Labeling protocol (15)3.3.1 Before you begin (15)3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations,and tissues (16)3.3.3 Labeling protocol for difficult tissue (17) (18)conversion3.4 Signal (19)4. Appendix (19)4.1 Trouble-shooting (22)4.2 References (23)4.3 Relatedproducts1.2 KitcontentsCaution The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.Avoid exposure and obtain special instructions before use.When using do not eat, drink or smoke. After contact with skin, washimmediately with plenty of water. In case of accident or if you feelunwell seek medical advice immediately (show label where possible).Collect the supernatants from the labeling reactions in a tightly closed,non-breakable container and indicate contents. Discard as regulatedfor toxic waste.Kit contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution•Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer•10× conc.•5×50l2 violet Label Solution•Nucleotide mixture in reaction buffer•1×conc.• 5 × 550 l3 yellow Converter-POD•Anti-fluorescein antibody, Fab frag-ment from sheep, conjugated withhorse-radish peroxidase (POD)•Ready-to-use• 3.5mlAdditional equipment required In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.Detailed information is given in front of each procedure.Procedure Equipment Reagents Preparation of sample material (section 3.2)•Adherent cells, cell smears and cytospinpreparations (section3.2.1)•Cryopreserved tissue (section 3.2.2.2)•Washing buffer: Phosphate buffered saline(PBS*)•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared •Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.2.1)•Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)•Washing buffer: PBS*•Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments•Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared •Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free•0.1 M Citrate buffer, pH 6 for microwave irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)•Micrococcal nuclease or •DNase I, grade I *Adherent cells, cell smears, cytospin preparations, and tissues (section 3.3.2) •Parafilm orcoverslips•HumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.3)•Plastic jar•Microwave•Humidifiedchamber•Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS*•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA*and 20% normal bovine serumSignal conversion (section 3.4)•Humidified chamber •Parafilm or coverslip •Washing buffer: PBS*•DAB Metal Enhanced Substrate Set* or alternative POD substrates •Mounting medium for light microscopy1.2 Kitcontents,continued2. Introduction2.1Product overviewTest principle Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Fig. 1: Test principleApplication The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:•Detection of individual apoptotic cells in frozen and formalin fixedtissue sections in basic research and routine pathology.•Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.•Typing of cells undergoing cell death in heterogeneous populationsby double staining procedures (6).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in latestages of necrosis (4, 38).DNA strand breaks may also be prominent in cell populations withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained. To confirm apoptotic mode of cell death, themorphology of respective cells should be examined very carefully.Morphological changes during apoptosis have a characteristic pattern.Therefore evaluation of cell morphology is an important parameter insituations where there is any ambiguity regarding interpretation ofresults.Sample material•Cytospin and cell smear preparations•Adherent cells cultured on chamber slides (31)•Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25,26, 29, 30, 32–34, 36, 39)Assay time2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections.Number of tests The kit is designed for 50 tests.Kit storage/ stability The unopened kit is stable at Ϫ15 to Ϫ25°C through the expiration date printed on the label.Reagent Storage and stabilityTUNEL reaction mixture The TUNEL reaction mixture should be pre-pared immediately before use and shouldnot be stored.Keep TUNEL reaction mixture on ice untiluse.Converter-POD Once thawed the Converter-POD solutionshould be stored at 2–8°C (maximum stabil-ity 6 months).Note: Do not freeze!Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at singlecell level at very early stages (1, 2, 6).Specific Preferential labeling of apoptosis versusnecrosis (3, 4).Fast Short assay time (2-3 h).Convenient•Reagents are provided in stable, opti-mized form.•No dilution steps required.Flexible•Suitable for fixed cells and tissue. Thisallows accumulation, storage and trans-port of samples (2, 5).•Double staining enables identification oftype and differentiation state of cellsundergoing apoptosis (6).Function-tested Every lot is function-tested on apoptoticcells in comparison to a master lot.2.2 BackgroundinformationCell death Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic pattern of structural changes in nucleus and cytoplasm,including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage tochromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant role in many pathological conditions like ischemia, stroke,heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).Identification of apoptosis Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:•Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4).•Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).•TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).2.2 Backgroundinformation,continued3. Procedures and required materialsThe working procedure described below has been developed andpublished by R. Sgonc and colleagues (6). The main advantage of thissimple and rapid procedure is the use of fluorescein-dUTP to labelDNA strand breaks. This allows the detection of DNA fragmentationby fluorescence microscopy directly after the TUNEL reaction priorto the addition of the secondary anti-fluorescein-POD-conjugate.3.1Flow chartAssay procedure The assay procedure is explained in the following flow chart.Adherent cells, cell smears and cytospin preparations Cryopreservedtissue sectionsParaffin-embeddedtissue sections↓↓↓Fixation •Dewaxation •Rehydration •ProteasetreatmentPermeabilisation of samples↓Addition of TUNEL reaction mixtureOPTIONAL: Analysis of samples by fluorescence microscopy↓Addition of Converter-PODAddition of Substrate solution↓Analysis of samples by light microscopy3.2Preparation of sample material3.2.1Adherent cells, cell smears and cytospin preparationsAdditional solutions required •Washing buffer: Phosphate buffered saline (PBS)•Blocking solution: 3% H2O2 in methanol•Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared•Permeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)Procedure In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at 15-25°C.2Rinse slides with PBS.3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).6Proceed as described under 3.3.3.2.2 Tissue sections3.2.2.1 Treatment of paraffin-embedded tissuePretreatment of paraffin embedded tissue Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!The other 3 alternative procedures are also described in the following table (step 2).Additional solutions required •Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water)•Washing buffer: PBS•Proteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 g/ml in 10 mM Tris/HCl, pH 7.4-8]Alternative treatments•Permeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly prepared•Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free•0.1 M Citrate buffer, pH 6 for the microwave irradiationProcedure In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g. by heating at 60°C followed by washing inxylene and rehydration through a graded series of ethanoland double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15-60 min at 37°C.3. Microwave irradiation •Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH6.0.•Apply 350 W microwave irradiation for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.2.1 Treatment of paraffin-embedded tissue, continued3.2.2.2Treatment of cryopreserved tissueAdditional solutions required •Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-pared•Washing buffer: PBS•Blocking solution: 3% H2O2 in methanol•Permeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue In the following table the pretreatment of cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at 15–25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at Ϫ15 to Ϫ25°C.3Incubate with Blocking solution for 10 min at 15–25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2–8°C).6Proceed as described under 3.3.3.3Labeling protocol 3.3.1Before you beginPreparation of TUNEL reaction mixtureOne pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) is sufficient for staining 10 samples by using 50 l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 l Label Solution per control.Note : The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture on ice until use.Additionalreagents required •Micrococcal nuclease or •DNase I, grade I (Cat. No. 104 132)ControlsTwo negative controls and a positive control should be included in each experimental set up.Step Action1Remove 100 l Label Solution (vial 2) for two negative con-trols.2Add total volume (50 l) of Enzyme solution (vial 1) to the remaining 450 l Label Solution in vial 2 to obtain 500 l TUNEL reaction mixture.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control:Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM T ris-HCl, pH 7.5, 10 mM MgCl 2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations andtissuesAdditional equipment and solutions required •Washing buffer: PBS •Humidified chamber •Parafilm or coverslipProcedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add50l TUNEL reaction mixture on sample.Note: For the negative control add 50 l Label solution each.To ensure a homogeneous spread of TUNEL reaction mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.5Rinse slide 3 times with PBS.6Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelengthin the range of 450–500 nm and detection in the range of515–565 nm (green).3.3.3 Labeling protocol for difficult tissueAdditional equipment and solutions required •Citrate buffer, 0.1 M, pH 6.0.•Washing buffer: PBS•Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum•Plastic jar•Microwave•Humidified chamberProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3•Apply 750 W (high) microwave irradiation for 1 min.•Cool rapidly by immediately adding 80 ml double dist.water (20–25°C).•Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!4Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 MpH 7.5, containing 3% BSA and 20% normal bovineserum.5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.6Add50µl of TUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in thedark.8•Rinse slide(s) three times in PBS for 5 min each.•Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in therange of 515–565 nm (green).3.4 SignalconversionAdditional equipment and solutions required •Washing buffer: PBS•Humidified chamber•Parafilm or coverslip•DAB Substrate* (Cat. No. 1 718 096) or alternative POD substrate •Mounting medium for light microscopyProcedure Please refer to the following table.Step Action1Dry area around sample.2Add50l Converter-POD (vial 3) on sample.Note: To ensure a homogeneous spread of Converter-PODacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or cover slip during incuba-tion.3Incubate slide in a humidified chamber for 30 min at 37°C.4Rinse slide 3× with PBS.5Add 50–100 l DAB Substrate or alternative POD substrates.6Incubate slide for 10 min at 15–25°C.7Rinse slide 3× with PBS.8Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.Alternative: Samples can be counterstained prior to analysisby light microscope.4. Appendix4.1 Trouble-shootingThis table describes various troubleshooting parameters. Problem Step/Reagent ofProcedurePossible cause RecommendationNonspecific labeling Embedding of tissue UV-irradiation forpolymerization ofembedding material(e.g. methacrylate)leads to DNA strandbreaksTry different embedding materialor different polymerizationreagent.Fixation Acidic fixatives (e.g.methacarn, Carnoy’sfixative)•Try 4% buffered paraformal-dehyde.•Try formalin or glutaralde-hyde.TUNEL reaction TdT concentration toohighReduce concentration of TdT bydiluting it 1:2 up to 1:10 withTUNEL Dilution Buffer (Cat. No.1 966 06).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-POD•Block with normal anti-sheepserum.•Block for 20 min with PBScontaining 3% BSA.•Reduce concentration ofconverter solution to 50%. Nucleases Some tissues (e.g.smooth muscles)show DNA strandbreaks very soon aftertissue preparation•Fix tissue immediately afterorgan preparation.•Perfuse fixative through livervein.Some enzymes arestill activeBlock with a solution containingddUTP and dATP.continued on next pageHigh back-ground Fixation Formalin fixation leadsto a yellowish stainingof cells containingmelanin precursorsTry methanol for fixation buttake into account that this mightlead to reduced sensitivity.TUNEL reaction Concentration oflabeling mix is toohigh for mamma car-cinomaReduce concentration of label-ing mix to 50% by diluting withTUNEL Dilution Buffer (Cat. No.1 966 006).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-POD•Block with normal anti-sheepserum.•Block for 20 min with PBScontaining 3% BSA.•Reduce concentration ofconverter solution to 50%. Sample Mycoplasma contami-nationMycoplasma detection Kit (Cat.No. 1 296 7449).Highly proliferatingcellsDouble staining e.g. withAnnexin-V-Fluos (Cat. No. 1 828681).Note: Measuring via microplatereader not possible because oftoo high background.Low labeling Fixation Ethanol and methanolcan lead to low label-ing (nucleosomes arenot cross-linked withproteins during fixa-tion and are lost dur-ing the proceduresteps)•Try 4% buffered paraformal-dehyde.•Try formalin or glutaralde-hyde.Extensive fixationleads to excessivecrosslinking of pro-teins•Reduce fixation time.•Try 2% buffered paraformal-dehyde.Permeabilisation Permeabilisation tooshort so that reagentscan’t reach their tar-get molecules•Increase incubation time.•Incubate at higher tempera-ture (e.g. 15–25°C).•Try Proteinase K (concentra-tion and time has to be opti-mized for each type oftissue).•Try 0.1 M sodium citrate at70°C for 30 min.continued on next pageProblem Step/Reagent ofProcedure Possible cause Recommendation4.1Trouble-shooting, continuedParaffin-embedding Accessibility forreagents is too low •Treat tissue sections afterdewaxing with Proteinase K (concentration, time andtemperature have to be opti-mized for each type of tis-sue).•Try microwave irradiation at370 W (low) for 5 min in200ml 0.1 M Citrate bufferpH 6.0 (has to be optimizedfor each type of tissue).No signal on positive control DNase treatment Concentration ofDNase is too low•For cryosections apply 3 U/mlDNase I, grade I.•For paraffin-embedded tissuesections apply 1500 U/mlDNase I, grade I.•In general, use 1 U/mlDNase I, grade I, dissolved in10 mM Tris-HCl pH 7.4 con-taining 10 mM NaCl, 5 mMMnCl2, 0.1 mM CaCl2, 25 mMKCl and incubate 30 min at37°C.•Alternative buffer 50 mMTris- HCl pH 7.5 containing1mM MgCl2 and 1 mg/mlBSA.Weak sig-nals Counterstaining Not suitable dye•Counterstaining with 5%methyl green in 0,1 M veronalacetate, pH 4.0 or Hematoxi-lin is possible (43).•Double-staining with propid-ium iodide is possible butonly for detection of morpho-logical cell changes.Problem Step/Reagent ofProcedure Possible cause Recommendation4.1Trouble-shooting, continued4.2 References1Gavrieli, Y., Sherman, Y. & Ben-Sasson, S. A. (1992) J. Cell Biol. 119, 493–501.2Gorczyca, W., Gong, J. & Darzynkiewicz, Z. (1993) Cancer Res. 53, 1945–1951.3Gorczyca, W. et al. (1993) Leukemi a 7, 659–670.4Gold, R. et al. (1994) Lab. Invest. 71, 219.5Gorczyca, W. et al. (1994) Cytometry 15, 169–175.6Sgonc, R. et al. (1994) Trends Genetics 10, 41–42.7Schmied, M. et al. (1993) Am. J. Pathol. 143, 446–452.8Wyllie, A. H. et al. (1980) Int. Rev. Cytol. 68, 251.9Kerr, J. F. R. et al. (1972) Br. J. Cancer 26, 239–257.10Duvall, E. & Wyllie, A. H. (1986) Immunol. To day 7, 115.11Compton, M. M. (1992) Canc. Metastasis Rev. 11, 105–119.12Allen, P. D., Bustin, S. A. & Newland, A. C. (1993) Blood Reviews 7, 63–73.13Cohen, J. J. & Duke, R. C. (1992) Annu. Rev. Immunol. 10, 267–293.14Clarke, P. G. H. (1990) Anat. Embryol. 181, 195–213.15Johnson, E. M. & Deckwerth, T. L. (1993) Annu. Rev. Neurosci. 16, 31–46.16Batistatou, A. & Greene, L. A. (1993) J. Cell Biol. 122, 523–532.17Strange, R. et al. (1992) Development 115, 49–58.18Carson, D. A. & Ribeiro, J. M. (1993) Lancet 341, 1251–1254.19Edgington, S. M. (1993) Biotechnology 11, 787–792.20Gougeon. M.-L. & Montagnier, L. (1993) Science 260, 1269–1270.21Hickman, J. A. (1992) Cancer Metastasis Rev. 11, 121–139.22Afanasyev, V. N. et al. (1993) Cytometry 14, 603–609.23Bryson, G. J., Harmon, B. V. & Collins, R. J. (1994) Immunology Cell Biology 72,35–4124Darzynkiewicz, Z. et al. (1992) Cytometry 13, 795–808.25Ando, K. et al. (1994) J. Immunol. 152, 3245–3253.26Berges, R. R. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8910– 8914.27Gorczyca, W. et al. (1992) Int. J. Oncol. 1, 639–648.28Gorczyca, W. et al. (1993) Exp. Cell Res. 207, 202–205.29Billig, H., Furuta, I. & Hsueh, A. J. W. (1994) Endocrinology 134, 245–252.30MacManus, J. P. et al. (1993) Neurosci. Lett. 164, 89–92.31Mochizuki, H. et al. (1994) Neurosci. Lett. 170, 191–194.32Oberhammer, F. et al. (1993) Hepatology 18, 1238–1246.33Portera-Cailliau, C. (1994) Proc. Natl. Acad. Sci. USA 91, 974 –978.34Preston, G. A. et al. (1994) Cancer Res. 54, 4214–4223.35Weller, M. et al. (1994) Eur. J. Immunol. 24, 1293–1300.36Zager, R.A. et al. (1994) J. Am. Soc. Nephrol. 4, 1588–1597.37Cohen, G. M. et al. (1992) Biochem. J. 286, 331–334.38Collins, R. J. et al. (1992) Int. J. Rad. Biol. 61, 451–453.39Sei, Y. et al. (1994) Neurosci. Lett. 171, 179–182.40Ansari, B. et al. (1993) J. Pathol. 170, 1–8.41Gold, R. et al. (1993) J. Histochem. Cytochem. 41, 1023–1030.42Negoescu, A. et.al. (1998) Biochemica3, 34-41.43Umermura, S. et al. (1996) J. Histochem. Cytochem. 44, 125-132 .。
罗氏TUNEL使用说明书(中文)
罗氏公司TUNEL细胞凋亡检测程序（In situ cell death detection kit-POD法）一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3'-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70％）、DAB工作液（临用前配制，5 µl 20×DAB＋1μL30％H2O2＋94 µl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl, pH 7.4-8）或细胞通透液（0.1% Triton X-100 in 0.1% sodium citrate，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH 7.5, 10 mM MgCl2，1 mg/ml BSA）等。
TUNEL说明书
不适用于诊断，仅供生命科学实验使用。
仅供体外使用。
TUNEL细胞凋亡原位检测试剂盒（标记POD）本试剂盒可标记DNA链末端，即TUNEL技术。
用于单个细胞水平凋亡（细胞程序性死亡）的免疫组织化学检测和定量分析。
光镜检测。
批号：11684817910一盒可用50次储存：-15—-25℃操作指南2006.01.1.2 试剂盒组成注意事项本品标记溶液含有二甲基砷酸盐，容易被吸入而产生毒性和导致呕吐；也含有二氯化钴，吸入后容易致癌。
因此应避免接触并遵循相关的操作说明。
使用过程中不能吃、喝和抽烟。
如果接触到皮肤，应立即用大量水冲洗干净。
如果感觉不适或者突发其它情况，应立即就医。
酶标反应应在致密的、无损坏的容器中进行，收集上清液后应标明成分。
垃圾应作为有毒废物进行处理。
注意：与先前的试剂盒/管不同，次试剂盒的酶溶解液不再含有毒的二甲砷基酸盐，因此瓶1没有毒性。
试剂盒组成请参照下表对比试剂盒组成试剂盒外自备仪器和试剂出上表所列试剂外，实验者需制备系列溶液。
下表列出了在不同实验步骤中所需要的试剂。
在每步操作前都给出了详细的说明。
2 引言2.1 产品描述实验原理在细胞凋亡过程中，基因组DNA 会断裂产生双链、低分子量的DNA 片段和高分子量的单链DNA 断端（缺口），这些DNA 链缺口可以利用酶标记核苷酸3’末端方法来识别。
应用原位细胞凋亡检测试剂盒具有准确、快速、简单、非辐射等特点，可以用来对细胞或者组织中的单个细胞进行检测并定量，因而次法被用在很多分析系统中，例如：●在基础研究和日常病理中检测冰冻和福尔马林固定的组织切片。
●在肿瘤研究和临床癌基因研究中确定某些恶性肿瘤对某种药物的敏感性。
●通过双染色操作，确定经历死亡的异常增生细胞的分型。
专一性TUNEL反应可以更好的标记通过凋亡产生的DNA链末端，因此就可以区分出凋亡与坏死，以及由抗肿瘤药物或者放射诱发的初始DNA链末端。
试验干扰假阴性：在某些形式的细胞凋亡中，DNA逃逸酶切或者不完全（37）。
tunel检测说明书
一、TUNEL试剂盒说明凯基一步法TUNEL细胞凋亡检测试剂盒(One Step TUNEL Apoptosis Assay Kit)为您提供了一种高灵敏度又快速简便的细胞凋亡检测方法，可检测细胞在凋亡过程中细胞核DNA的断裂情况，其原理是绿色荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下,可以连接到凋亡细胞中断裂的DNA的3'－OH末端，可用荧光显微镜检测。
由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-OH形成，很少能够被标记。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）的凋亡原位检测。
对于经过固定和洗涤的细胞或组织，只要经过一步染色反应，洗涤后就可以通过荧光显微镜检测到凋亡细胞。
本试剂盒特点●操作简便：使用Ready-to-Use型试剂，只需一步染色反应，洗涤后即可观察。
●高灵敏度：可以单一检出初期的凋亡细胞。
●高特异性：能特异性染色凋亡细胞。
●快速操作：仅需约1-2个小时即可完成整体操作。
●方便观察：可使用荧光显微镜观察实验结果。
●高正确性：有阳性对照片的制备方法，可以确认试剂盒的有效性●应用范围广：可以用于冷冻或石蜡切片中的组织细胞凋亡检测，也可检测培养的贴壁细胞或悬浮细胞的凋亡情况。
二、TUNEL试剂盒组分注意事项1．使用前请认真阅读本说明书，提前准备好相关试剂。
2．因本试剂盒中组分均为微量，使用前请离心集液。
3．为避免试验误差、降低试剂的损耗，建议使用精密度高的进口微量移液枪及枪头。
4． TdT 酶反应液最好在使用前根椐样本数量集中配制，再分别滴加于各样本片上，避免每个样本单独配制而产生的试剂损耗。
5. 用户自备：二甲苯、多聚甲醛、PBS、H2O2、TritonX-100、柠檬酸钠、盖玻片、载玻片、染色缸。
三、操作流程概览细胞涂片、贴壁细胞爬片等细胞样本四、检测样本的预处理TUNEL检测时样本的预处理是试验的关键所在，本说明书推荐的条件仅为普遍情况，用户需根椐自已的样本材料及首次试验结果来调整各个条件（参照Page 10），如处理时间、处理浓度等，来优化出适合自身样本的试验条件，从而做出客观的试验结果。
TUNEL细胞凋亡原位检测试剂盒说明书
TUNEL细胞凋亡原位检测试剂盒(通用)（BIOTIN标记POD法,适用于细胞、组织样本）一 TUNEL检测原理凯基TUNEL细胞凋亡检测试剂盒是用来检测细胞在凋亡过程中细胞核DNA的断裂情况，其原理是生物素（biotin）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下,可以连接到凋亡细胞中断裂的DNA的3‘－OH末端，并可与连接了的辣根过氧化酶的链霉亲和素（Streptavidin-HRP）特异结合，在辣根过氧化酶底物二氨基联苯胺（DAB）的存在下，产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在普通显微镜下即可观察和计数凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）的凋亡原位检测。
二 TUNEL试剂盒组分组份20 assays 50 assays 100 assays 储存条件Equilibration Buffer 1.0 mL 2.5 mL 5.0 mL -20℃Biotin-11-dUTP 20μL 50μL 100μL -20℃TdT Enzyme 80μL 200μL 400μL -20℃50×Proteinase K40μL 100μL 200μL -20℃Streptavidin-HRP 10μL 25μL 50μL 4℃避光DAB 2mg 5mg 10 mg -20℃注意事项1 使用前请认真阅读本说明书，提前准备好相关试剂。
2 因本试剂盒中组分均为微量，使用前请离心集液。
3 为避免试验误差、降低试剂的损耗，建议使用精密度高的进口微量移液枪及枪头。
4 TdT 酶反应液最好在使用前根椐样本数量集中配制，再分别滴加于各样本片上，避免每个样本单独配制而产生的试剂损耗。
5 另因DAB为固体粉末，使用前加入适量PBS溶解，配制成20×DAB（10 mg/ml）后，按下述方法显色使用。
TUNEL细胞凋亡试剂盒内容及操作步骤
TUNEL细胞凋亡试剂盒内容及操作步骤一、试剂盒内容2.终止缓冲液：用于停止DNA链的合成反应，防止假阳性结果的产生。
3.蛋白酶K：用于溶解细胞质膜和核膜，使DNA暴露出来。
4.异硫氰酸荧光素（FITC）标记转移酶：用于检测dUTP与DNA标记的连接。
5.正控组织切片或细胞悬液：用于检验试剂盒的敏感性和特异性。
二、操作步骤1.细胞处理将要测试的细胞分成实验组和对照组，分别处理。
实验组是要测试凋亡的细胞，对照组是不会凋亡的细胞。
2.固定细胞用4%的乙醛或无氧乙醛等适当浓度的固定液固定细胞。
涂片上的细胞需要进行细胞穿孔，可以使用0.1%的Triton X-100进行细胞穿孔。
3.蛋白酶K消化将蛋白酶K溶解在PBS缓冲液中，加入溶液中胶原酶和蛋白酶蚀解，接着加入盛有细胞的离心管中。
将离心管放入37℃恒温水浴中，反应20-30分钟。
4.清洗细胞用PBS缓冲液洗涤细胞3次。
每次用PBS缓冲液冲洗细胞5分钟。
5.脱水将细胞离心，抽去PBS缓冲液，然后使用3%的过氧化氢来进行脱水。
6.TUNEL染色将细胞加入等量的TUNEL试剂液，轻轻混合后，将离心管放入37℃恒温水浴中，反应30-60分钟。
7.洗涤细胞用PBS缓冲液洗涤细胞3次。
每次用PBS缓冲液冲洗细胞5分钟。
8.反应终止使用终止缓冲液反应终止DNA链合成反应，将离心管放入室温中静置10分钟。
9.洗涤细胞用PBS缓冲液洗涤细胞3次。
每次用PBS缓冲液冲洗细胞5分钟。
10.展片与观察将细胞滴在载玻片上，并加入封片缓冲液。
在载玻片上覆盖盖玻片，用显微镜观察。
通过上述步骤，可以使用TUNEL细胞凋亡试剂盒对细胞进行凋亡分析。
该试剂盒可根据DNA末端的3'-OH羟基与终止缓冲液中标记的dUTP之间的连接，用显微镜观察细胞内是否存在凋亡现象。
荧光标记的dUTP可通过荧光显微镜直接观察，而酶标记的dUTP则需要加入相应的底物，通过酶的催化反应后可通过光学密度法或荧光显微镜来定量测定。
翻译好的 罗氏公司Tunel试剂盒操作说明方案 (2)
罗氏(R o c h e)公司T u n e l试剂盒操作说明书(Insitucelldeathdetectionkit-POD法)一、原理：TUNEL（TdT-mediateddUTPnickendlabeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdTEnzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radishperoxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）EnzymeSolution酶溶液：TdT10×、2号（紫盖）LabelSolution标记液：荧光素标记的dUTP1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5μl20×DAB+1μL30%H2O2+94μlPBS）、ProteinaseK工作液（10-20μg/mlin10mMTris/HCl，pH7.4-8）或细胞通透液（0.1%TritonX-100溶于0.1%柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase1（3000U/ml–3U/mlin50mMTris-HCl，pH7.5，10mMMgCl2，1mg/mlBSA）等。
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罗氏tunel检测细胞凋亡试剂盒说明书
800-820-0577
11684817910
注意：Label溶液含有甲次砷酸盐和二氯化钴，严禁吸入和食入。
反应悬浮物收集于密闭、不易碎、有明确标识的容器中，按有毒废物处理。
需要自己配置的其他物品：
除上表所列试剂外，还需准备以下溶液。
下表列出每步所需物品概览：
产品概述：
特异性：TUNEL反应优先标记凋亡产生的DNA链断裂，从而辨别凋亡与坏死、以及由抑制细胞生长的药物或放射线产生的primary DNA链断裂
实验干扰：假阴性：在某些型式的凋亡细胞中DNA链断裂可能缺失或不完全。
空间位阻，如细胞外元件可能阻止TdT到达DNA断裂处。
两种情况均能产生假阴性。
假阳性：在坏死晚期，可能产生大量的DNA片段
DNA链断裂也可能在具有高增殖和代谢活动的细胞中出现。
两种情况均能产生
假阳性。
为确认细胞死亡的凋亡型式，应认真进行每种细胞的形态学检查
凋亡过程中产生的形态学改变尤其特征形式，因此，对于可以结果进行解释时，
细胞形态评估是一项重要的参数
样本：细胞离心涂片和细胞涂片
在chamber slides上培养的黏附细胞
冰冻或福尔马林固定、石蜡包埋样本
分析时间：2-3小时，除外培养、固定和渗透
检测次数：一个试剂盒50T
试剂盒存储/稳定性：未开封试剂盒储存于-15～-25℃可稳定至标签上标明的效期。
步骤和所需材料：
1 流程图：
2 样品准备
2.1 黏附细胞、细胞涂片和细胞离心涂片
需准备的其他试剂：Washing buffer：磷酸盐缓冲液（PBS）
Blocking buffer封闭溶液：甲醇稀释的3% H2O2
Fixation solution固定溶液：PBS配制的4%多聚甲醛，ph 7.4，新鲜配制
Permeabilisation solution 渗透液：0.1%Triton1）X-100溶于0.1%柠檬酸钠
溶液中，新鲜配制
步骤：下表描述了细胞固定、内源性过氧化物酶封闭和细胞渗透过程。
注意：固定和渗透另外两个细胞样本用于银杏和阳性对照
2.2 组织部分
2.2.1 福尔马林-包埋组织
福尔马林包埋组织的预处理：可按4种不同的方式预处理。
如用蛋白酶K，不含核酸酶，浓
度、孵育时间和温度应按组织类型优化
注意：只用罗氏应用科学的蛋白酶K，因其经检测不含核酸酶，
核酸酶可导致假阳性。
另外3中替代方法在下表中描述（step 2）
需准备的其他试剂：二甲苯和乙醇（浓度：95%，90%，80%，70%，溶于双蒸水中）
Washing buffer：PB S
蛋白酶K，不含核酸酶，工作浓度：[10-20ug/ml，溶于10mM Tris/HCL
中，ph7.4-8]
替代处理方案
※渗透溶液：0.1%Triton1）X-100，溶于0.1%柠檬酸钠的，新鲜配制
※胃蛋白酶*（0.25%-0.5%，溶于盐酸中，ph2）或胰蛋白酶*，0.01N HCL，
不含核酸酶
※0.1M柠檬酸缓冲液，ph6，微波照射
步骤：下表描述了用蛋白酶K（不含核酸酶）和3中替代方法预处理福尔马林-包埋组织的方法
2.2.2 冰冻组织处理（略）
3 标记草案
3.1 开始之前
准备TUNEL反应混合液：每一对管子（小瓶1：酶溶液，小瓶2：标记溶液）足以用于50ul
反应体系的10张片子，和2个50ul标记溶液的阴性对照。
注意：TUNEL反应混合液应于用前临时配制，不能储存。
TUNEL
反应混合液用前置于冰上
需准备的其他试剂：微球菌核酸酶或
DNase I，重组，级别1*
对照：每次实验应包含两个阴性对照和一个阳性对照应
3.2 黏附细胞、细胞涂片、细胞离心涂片和组织标记草案
需准备的其他设备和试剂：Washing buffer：PBS
湿盒
Parafilm石蜡封口膜或盖片
步骤：参考下表
3.3 困难组织标记草案（略）
3.4 信号转换
需要准备的其他设备和试剂：Washing buffer：PBS
湿盒
Parafilm石蜡封口膜或盖片
DABA底物或POD替代底物
光镜镜检封固剂
步骤：按照下表操作
4. 附件：。