关于 oct4 ssea4 tra-81-1的免疫荧光染色问题

maxiaorongqp

各位大虾  ，OCT4 免疫荧光染色前 用TRITON 处理多长时间比较合适？ SSEA4 和TRA-81-1 是否需要用TRITON 处理 ？

饶冠华

我没有做过oct4的染色，但问过师姐 说 先用0.2%triton 打孔10分钟 冰上，然后再用1M HCl RT 10min----2M HCl 37°C 20min  ----PBS 3*10min

xyzengh

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, J9 _0 F6 n; K! R  V* U( b& D0 t) LOct4是核抗原，所以triton是必须的，SSEA3/4是膜表面抗原，可加可不加，你为了方便可以都加，把我文章里面的方法发上来给你参考吧。/ s& r8 u, }5 x& n6 j# t
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Immunofluorescence Analysis$ m) f- J3 m; c* D! l: j
Cells were fixed with 4% paraformaldehyde for 10 min., incubated in PBS containing 5% rabbit serum and 0.4% Triton X-100 for blocking and permeablization, respectively. PBS containing 0.5% Tween 20 (PBS-T) was used to dilute antibodies and wash the cells in the following procedures. The cells were incubated with antibodies against OCT4, SSEA-4，TRA-1-60 and TRA-1-81 at 37 °C for 1 h, followed by washing with PBS-T for three times. Afterwards, the cells were incubated with fluorochrome-conjugated, corresponding secondary antibodies at 37 °C for 45 min. and washed with PBS-T for three times. Finally, the cells were examined under fluorescence microscope to capture both phase and fluorescent images.

xyzengh

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  M+ Z9 D3 W2 a再附上几张图让你有个感性认识吧。

maxiaorongqp

谢谢  请问你用0.4%的triton-100破膜 多久

xyzengh

10分钟就足够了

cortex

这个与你要染的部位有关，细胞膜上可以不透膜，胞质和胞核里的时间是不一致的。一般15－30min。

hcluo

是用激光共聚焦做的吗？

xyzengh

不是，普通的荧光显微镜就行了。

sniper

20_30min 就行，你也可以做几个不同时间处理的组对比一下