求救：免疫荧光染色中抗体的选择

qgjin

免疫荧光染色中 所选择的一抗，二抗 以及封闭液中serum 他们之间的关系0 |. x9 \/ Y7 Y/ X7 J* l  V( d
再者就是： 怎样选择 上述试剂 才能染成最佳的效果？

枕头和书

抗体买来之后要检测看效果，效果好的按要求保存都没什么问题。
7 Y! f; Y4 z; p  X, {. b% d封闭液又几种配方看做什么了。通用的这几个都很好用都能做的出来。0 g9 b& v# v4 V# o0 d5 q- q
关键是实验操作时注意时间的把握和洗脱时间次数。、
; O! X9 }/ G2 R* |; S! r楼主要做哪方面的实验

qgjin

回复 2# 枕头和书
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8 X6 m1 _. _7 [我想做精原干细胞的免疫荧光染色 但对抗体的 选择和serum的选择上有点左右不定，因此来这里请教一下

qgjin

1. For confocal laser microscope observation, Isolation cells were plated on a chamber slide with MEF feeders.
. t3 s) g! w2 p& h  2. Cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.
4 M+ R. F% t5 C6 H  3.  Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min. ' q" z+ d' a+ m* R" U/ K
步骤3后 要不要用PBS 清洗后进行以下步骤？
- i# \" l5 L4 W' ]3 O3 H3 W  4. Then cells were blocked with 5% bovine serum albumin in PBS for 1 h.
9 R: k3 c; x: M" F  5. The cells were incubated with primary antibodies for 16 h at 4 ° C. （过夜？）
: Q1 T9 j: f" M) L* T 6.  The Cells were washed twice in PBS 。
1 T9 K4 h, S' P. E1 _4 e  7. Then the cells were incubated with secondary antibodies for 1 h at 37 ° C.
) t: M& B/ k  Y7 X$ F  8.  For nuclear staining, the cells were stained with Hoechst 33,258 for 15 min at room temperature.
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请大侠 批评改正

lixiaodong

封闭液如用serum，应用二抗宿主来源的serum，不能用一抗宿主的serum。

asssan

细胞固定用-20度甲醇，可以不需破膜