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
. a2 B+ x+ p  R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。5 E# Y' x2 `3 Y& u, |0 l/ Q
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。9 [# ^2 s' w" r# y' W% u
关键是实验操作时注意时间的把握和洗脱时间次数。、
* b& {8 Q# H" e/ x7 i( {楼主要做哪方面的实验

qgjin

回复 2# 枕头和书
( f0 R+ K$ ?0 b2 I3 B  _
) G7 A0 o% N# i8 d8 y% S% ~& B' k- z# L  ?" C8 `/ l: I7 \
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.
. \4 m8 I. U: J5 w* Z  2. Cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.
& e7 K; o  O9 B. _+ W: m$ R  3.  Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min.
& \4 r6 o0 o$ }% o& A步骤3后 要不要用PBS 清洗后进行以下步骤？
# D' p" |& [1 n% c& K) g/ j  4. Then cells were blocked with 5% bovine serum albumin in PBS for 1 h. 4 V) ]6 t0 ~$ X! u) K' C
  5. The cells were incubated with primary antibodies for 16 h at 4 ° C. （过夜？）1 a  d% V% b, b) K2 w
6.  The Cells were washed twice in PBS 。" R2 o- c: V+ b" j6 Y1 q0 S; V
  7. Then the cells were incubated with secondary antibodies for 1 h at 37 ° C.
( Q# h# J6 @: e  8.  For nuclear staining, the cells were stained with Hoechst 33,258 for 15 min at room temperature.9 ]* j4 y8 I2 g# ?% H1 ?# v+ V2 o

& a; N+ j5 d8 m3 n6 g" x$ d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