[Histonet] Re: Immunocytochemistry for Systems biology

[Michelle MacVeigh-Aloni]

Hi Vinay,

I am not an expert in this field... 
We use this protocol for frozen sections and it works. For cells grown on coverslips you skip step #6.

Try this :

Fix

1. PBS                                     2 x 3min

 

2. NH4Cl                                 5 min           (50mM in PBS) (Removes the autofluorescence from the aldehyde groups of the fixative)

 

3. PBS                                     2 x 3 min

 

4. 0.1% Tx-100                           5 min (Permeabilises the cell walls)

 

5. PBS                                     2 x 3 min

 

6. SDS                                     5 min          (1% SDS in PBS) (works like antigen retrieval)

 

7. PBS                                     3 x 5 min

 

8. Block  1% BSA in PBS    10 min or longer



9. Primary Ab                        40min in 37oC or 1 to 2 hours at room temp

 

10. PBS                                  2 x 5 min

 

11. Secondary Ab                  40min in 37oC

 

12. PBS                                  2 x 5 min

 

13. Coverslip with Anti fade medium

      Let dry overnight



Good luck with it



Michelle

Research specialist

USC Kecl School of Medicine