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
  ]& a3 M7 [% I# ?* Q+ V' G# t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。  L3 L4 Z$ D4 W# g
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。
4 I& ?1 Q* |  ^5 i* F: n关键是实验操作时注意时间的把握和洗脱时间次数。、
7 @: _+ i4 K! q# z; T楼主要做哪方面的实验

qgjin

回复 2# 枕头和书
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6 ^' }2 @! g+ C0 }4 s+ z" s3 ]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.3 X+ d3 F, E+ E0 u
  2. Cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.- v: _- c7 [8 N% m2 r7 f% I) i: m
  3.  Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min. : b, ^! M% n% B3 z& s$ \# K; R$ D
步骤3后 要不要用PBS 清洗后进行以下步骤？  P0 A8 g& G: m# R0 C. L
  4. Then cells were blocked with 5% bovine serum albumin in PBS for 1 h. 9 E2 J# W6 |9 l9 |* X2 W7 p( E
  5. The cells were incubated with primary antibodies for 16 h at 4 ° C. （过夜？）
; H, r+ ?" q4 z( ^( Z/ w& E- S) g 6.  The Cells were washed twice in PBS 。1 J( h, {' x% j# m. u9 n7 d' i
  7. Then the cells were incubated with secondary antibodies for 1 h at 37 ° C.5 J6 v1 _9 t4 W5 U: D$ E
  8.  For nuclear staining, the cells were stained with Hoechst 33,258 for 15 min at room temperature.
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4 {- w4 K: X- h8 R4 G* K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