常用荧光探针小结
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14
七、Hoechst
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by Hoechst AG. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra.
12
A type of simple fluorescence: DAPI is excited in near UV with 365 nm and emitted in the violet-blue spectral range. The marking reveals cell nuclei and particularly the chromosomes
3
二、荧光素酯
酯酶
Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) is a cell permeable dye generally used in animal cell proliferation research. CFDASE enters cells by diffusion and is cleaved by intracellular esterase enzymes to form an amine-reactive product, CFSE. This product produces a detectable fluorescence and covalently binds to intracellular lysine residues and other amine sources.
15
16
Hoechst 33258 (magenta) bound to the minor groove of DNA (green and blue)
Transmission image of HeLa cells, with overlay of Hoechst 33258 staining (blue).
7
四、罗丹明200
AC41323-0010
RB200，也称丽丝胺罗丹明B 无定形褐红色粉末，不溶于水，易溶于酒精 和丙酮，性质稳定，可长期保存，最大吸收光谱为570nm，呈明亮的橙色荧 光，因与FITC的黄绿色有明显区别，故被广泛用于对比染色或用于两种不同 颜色的荧光抗体的双重染色。
标 记 方 法 方法：取1g RB200及五氯化磷（PCL5）2g放乳钵中研磨5min （在毒气操作橱中），加10ml无水丙酮，放置5min，随时搅拌，过滤，用所 得溶液进行结合。将每亳升血清用1ml生理盐水及1ml碳酸盐缓冲液 （0.5mol/L，pH9.5）稀释，逐滴加入0.1ml RB200溶液，随加随搅拌，在04℃继续搅拌 12-18h。
4
视频：The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
5
三、异硫氰酸罗丹明（TMRITC）
四甲基异硫氰酸罗达明，它是一种紫红色粉末，较稳定。其最 大吸收光谱为550nm，最大发射光谱620nm，呈橙红色荧光，与FITC 的黄绿色荧光对比清晰，与蛋白质结合方式同TITC。它可用于双标 记示踪研究。
1
Baidu Nhomakorabea
Exitation λmax: 495 nm; Emission λmax: 519 nm; Solvent pH:8.00
2
Immunocytochemistry/Immunofluorescence-alpha Tubulin antibody [DM1A] (FITC) (ab64503)
13
Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubles are marked green by an antibody and actin filaments are labelled red with phalloidin.
ICC/IF image of ab64503 stained human HeLa cells. The cells were methanol fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab64503, 1µg/ml, FITC conjugated (green)) for 1h at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
8
Confocal image of double immunostaining for Akt in hippocampal CA1 pyramidal neurons. Section is shown from a normal rat. The red color derived from lissamine rhodamine conjugated secondary antibody represents MAP2, the green color derived from fluorescein indicates the labeling of Akt. Yellow represents overlay of red and green.
17
八、碘化丙啶(Propidium Iodide, PI）
PI
EB
Propidium iodide is an intercalating agent and a fluorescent molecule that can be used to stain cells. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm. Excitation energy can be supplied with a xenon or mercury-arc lamp or with the 488 line of an argon-ion laser. Propidium iodide is used as a DNA stain for both flow cytometry, to evaluate cell viability or DNA content in cell cycle analysis, and microscopy to visualise the nucleus and other DNA containing organelles. It can be used to differentiate necrotic, apoptotic and normal cells.
11
DAPI (magenta) bound to the minor groove of DNA (green and blue).
DAPI can be used for fixed cell staining, the concentration of DAPI needed for live cell staining is generally very high and rarely used for live cells. Though it was not shown to have mutagenicity to E. coli, it is labelled as a known mutagen in manufacturer information. As it is a DNA binding compound it is likely to have some low level mutagenic properties and care should be taken in its handling and disposal.
DAPI is a dye which binds to DNA and shows an increased blue fluorescence when bound to DNA. The blue fluorescent spots in the picture show the nuclei in the mycel.
常用荧光探针小结
一、异硫氰酸荧光素（Fluorescein isothiocyanate, FITC） FITC有两种异构体，性质稳定，低温下干燥保存，其
性状多年不变，室温下也能保存两年以上。异构体I、II均 能与蛋白质良好结合，但异构体I的荧光效率更高，与蛋白 质的结合也更稳定。 FITC的最大吸收光谱为490----495纳 米，最大发射光谱为520-530nm，呈明亮的黄绿色荧光。 FITC含有异硫氰基 ， 在碱性条件下能与IgG的自由氨基 （主要是赖氨酸的-氨基）形成荧光抗体结合物。
6
Detection of α-tubulin in A549 cells demonstrates use of rhodamine-labeled secondary antibody. Cells were probed with a mouse anti-α-tubulin primary antibody (0.4µg/mL) and Rhodamine-goat anti-mouse secondary antibody (2µg/mL). Nuclei were labeled with Hoechst Dye. Images were acquired by fluorescence microscopy. A. Fluorescence image shows a delicate network of α-tubulin (pseudo-colored green) located exclusively in the cytoplasm. B. Nuclear counterstain with Hoechst Dye (pseudo-colored blue) C. Merged image.
9
五、溴化乙锭
详见第四节“应用于核酸检测的荧光探针技术”
10
六、DAPI （ 4‘,6-diamidino-2-phenylindole）
DAPI was first synthesised in as part of a search for drugs to treat trypanosomiasis. Although it was unsuccessful as a drug, further investigation indicated it bound strongly to DNA and became more fluorescent when bound. When bound to double-stranded DNA DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore for fluorescence microscopy DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.
七、Hoechst
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by Hoechst AG. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra.
12
A type of simple fluorescence: DAPI is excited in near UV with 365 nm and emitted in the violet-blue spectral range. The marking reveals cell nuclei and particularly the chromosomes
3
二、荧光素酯
酯酶
Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) is a cell permeable dye generally used in animal cell proliferation research. CFDASE enters cells by diffusion and is cleaved by intracellular esterase enzymes to form an amine-reactive product, CFSE. This product produces a detectable fluorescence and covalently binds to intracellular lysine residues and other amine sources.
15
16
Hoechst 33258 (magenta) bound to the minor groove of DNA (green and blue)
Transmission image of HeLa cells, with overlay of Hoechst 33258 staining (blue).
7
四、罗丹明200
AC41323-0010
RB200，也称丽丝胺罗丹明B 无定形褐红色粉末，不溶于水，易溶于酒精 和丙酮，性质稳定，可长期保存，最大吸收光谱为570nm，呈明亮的橙色荧 光，因与FITC的黄绿色有明显区别，故被广泛用于对比染色或用于两种不同 颜色的荧光抗体的双重染色。
标 记 方 法 方法：取1g RB200及五氯化磷（PCL5）2g放乳钵中研磨5min （在毒气操作橱中），加10ml无水丙酮，放置5min，随时搅拌，过滤，用所 得溶液进行结合。将每亳升血清用1ml生理盐水及1ml碳酸盐缓冲液 （0.5mol/L，pH9.5）稀释，逐滴加入0.1ml RB200溶液，随加随搅拌，在04℃继续搅拌 12-18h。
4
视频：The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
5
三、异硫氰酸罗丹明（TMRITC）
四甲基异硫氰酸罗达明，它是一种紫红色粉末，较稳定。其最 大吸收光谱为550nm，最大发射光谱620nm，呈橙红色荧光，与FITC 的黄绿色荧光对比清晰，与蛋白质结合方式同TITC。它可用于双标 记示踪研究。
1
Baidu Nhomakorabea
Exitation λmax: 495 nm; Emission λmax: 519 nm; Solvent pH:8.00
2
Immunocytochemistry/Immunofluorescence-alpha Tubulin antibody [DM1A] (FITC) (ab64503)
13
Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubles are marked green by an antibody and actin filaments are labelled red with phalloidin.
ICC/IF image of ab64503 stained human HeLa cells. The cells were methanol fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab64503, 1µg/ml, FITC conjugated (green)) for 1h at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
8
Confocal image of double immunostaining for Akt in hippocampal CA1 pyramidal neurons. Section is shown from a normal rat. The red color derived from lissamine rhodamine conjugated secondary antibody represents MAP2, the green color derived from fluorescein indicates the labeling of Akt. Yellow represents overlay of red and green.
17
八、碘化丙啶(Propidium Iodide, PI）
PI
EB
Propidium iodide is an intercalating agent and a fluorescent molecule that can be used to stain cells. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm. Excitation energy can be supplied with a xenon or mercury-arc lamp or with the 488 line of an argon-ion laser. Propidium iodide is used as a DNA stain for both flow cytometry, to evaluate cell viability or DNA content in cell cycle analysis, and microscopy to visualise the nucleus and other DNA containing organelles. It can be used to differentiate necrotic, apoptotic and normal cells.
11
DAPI (magenta) bound to the minor groove of DNA (green and blue).
DAPI can be used for fixed cell staining, the concentration of DAPI needed for live cell staining is generally very high and rarely used for live cells. Though it was not shown to have mutagenicity to E. coli, it is labelled as a known mutagen in manufacturer information. As it is a DNA binding compound it is likely to have some low level mutagenic properties and care should be taken in its handling and disposal.
DAPI is a dye which binds to DNA and shows an increased blue fluorescence when bound to DNA. The blue fluorescent spots in the picture show the nuclei in the mycel.
常用荧光探针小结
一、异硫氰酸荧光素（Fluorescein isothiocyanate, FITC） FITC有两种异构体，性质稳定，低温下干燥保存，其
性状多年不变，室温下也能保存两年以上。异构体I、II均 能与蛋白质良好结合，但异构体I的荧光效率更高，与蛋白 质的结合也更稳定。 FITC的最大吸收光谱为490----495纳 米，最大发射光谱为520-530nm，呈明亮的黄绿色荧光。 FITC含有异硫氰基 ， 在碱性条件下能与IgG的自由氨基 （主要是赖氨酸的-氨基）形成荧光抗体结合物。
6
Detection of α-tubulin in A549 cells demonstrates use of rhodamine-labeled secondary antibody. Cells were probed with a mouse anti-α-tubulin primary antibody (0.4µg/mL) and Rhodamine-goat anti-mouse secondary antibody (2µg/mL). Nuclei were labeled with Hoechst Dye. Images were acquired by fluorescence microscopy. A. Fluorescence image shows a delicate network of α-tubulin (pseudo-colored green) located exclusively in the cytoplasm. B. Nuclear counterstain with Hoechst Dye (pseudo-colored blue) C. Merged image.
9
五、溴化乙锭
详见第四节“应用于核酸检测的荧光探针技术”
10
六、DAPI （ 4‘,6-diamidino-2-phenylindole）
DAPI was first synthesised in as part of a search for drugs to treat trypanosomiasis. Although it was unsuccessful as a drug, further investigation indicated it bound strongly to DNA and became more fluorescent when bound. When bound to double-stranded DNA DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore for fluorescence microscopy DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.