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; G) Y1 J6 @  d
再者就是： 怎样选择 上述试剂 才能染成最佳的效果？

qgjin

回复 2# 枕头和书 . S6 _1 Q2 r  w+ H  q

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& i1 s& T- e, k- Q5 P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 J0 X' l6 r7 k% |  2. Cells were washed twice in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature.
# u4 l  {! B2 m" @1 s) ]5 p8 q, {  ~  3.  Fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min.
. ^2 |  {( @7 A步骤3后 要不要用PBS 清洗后进行以下步骤？
0 C0 l2 U  f3 v  E7 t* y9 J; S# O  Z4 @  4. Then cells were blocked with 5% bovine serum albumin in PBS for 1 h.   S+ E/ t( Z) z/ A- N# b
  5. The cells were incubated with primary antibodies for 16 h at 4 ° C. （过夜？）
  R" h8 J" {' Q& O 6.  The Cells were washed twice in PBS 。
7 c! ?: X7 \; ]8 r7 E3 h' W& H  7. Then the cells were incubated with secondary antibodies for 1 h at 37 ° C.: ]/ O0 b, B+ K1 _5 W3 b
  8.  For nuclear staining, the cells were stained with Hoechst 33,258 for 15 min at room temperature.2 G' }0 e4 f2 R- K! v+ x# ^

5 N: |' l2 O+ Q0 a' l请大侠 批评改正