Methods and Techniques Discussion => Antigen Retrieval => Topic started by: SCristina on January 24, 2014, 11:38:15 AM

Title: Problems with antigen exposition in retinal cells
Post by: SCristina on January 24, 2014, 11:38:15 AM
Our group are working with retinal tissue fixed with 4% PFA. 

We did antigen retreival with citrate sodium ph6 with a pressure cooker during 3 minuts.
The immunostaning was against GFAP (1:200). We also used 10% normal goat serum and 0,5% BSA as a blocking solution.

The problem was that the staining was really low. Any suggestion? Any permeabilization step could be positive? 


Thanks!   :)     
Title: Re: Problems with antigen exposition in retinal cells
Post by: gula on January 25, 2014, 09:35:20 AM
You can prolong your antigen retrieval. Try serial sections and do HIER with 5 to 15 min.
If your tissue is properly fixed, it should withstand the procedure.
Important: adhesive slides, cooling step after HIER for 10-15 min.

gula