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
  `8 H- B. k. v* Q& W% t1 S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。1 h' _0 Z' b6 F$ v
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。: J4 i4 d) E  y+ r
关键是实验操作时注意时间的把握和洗脱时间次数。、- D, k2 s) T5 l6 n4 j! I
楼主要做哪方面的实验

qgjin

回复 2# 枕头和书
5 B, l8 e4 y' ?+ a4 L9 a2 y) ]3 P$ b+ o+ l
1 ]& J5 Y+ {6 Y4 Q7 z2 C: ?8 t: x9 q/ l
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.
% U3 a- J7 X% Y, w" o  2. Cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.$ g" ]4 U  w  {9 q  {% c3 J
  3.  Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min. 6 i8 H1 T5 C" C. U' [
步骤3后 要不要用PBS 清洗后进行以下步骤？, f( c* L$ D2 ?1 L
  4. Then cells were blocked with 5% bovine serum albumin in PBS for 1 h.
) ?+ K% T& x! b7 Q: @  5. The cells were incubated with primary antibodies for 16 h at 4 ° C. （过夜？）
& U& a- u" m4 Q: L+ Y7 O 6.  The Cells were washed twice in PBS 。" q- \0 C, ?/ |+ }2 F4 z7 N
  7. Then the cells were incubated with secondary antibodies for 1 h at 37 ° C.
- R& Y, \4 U1 w- \- n/ N  8.  For nuclear staining, the cells were stained with Hoechst 33,258 for 15 min at room temperature." D: {! T7 ^) o' O4 ~* |

  s7 }1 I' G- Q1 X+ n% W( j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