常用荧光探针小结上课讲义
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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。
3源自文库
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.
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).
1
Exitation λmax: 495 nm; Emission λmax: 519 nm; Solvent pH:8.00
2
Immunocytochemistry/Immunofluorescence-alpha Tubulin antibody [DM1A] (FITC) (ab64503)
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。
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.
7
四、罗丹明200
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。
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.
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。
3源自文库
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.
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).
1
Exitation λmax: 495 nm; Emission λmax: 519 nm; Solvent pH:8.00
2
Immunocytochemistry/Immunofluorescence-alpha Tubulin antibody [DM1A] (FITC) (ab64503)
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。
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.
7
四、罗丹明200
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。
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.