We are doing mice retina flatmounts on AAV experiments. I want to use an antirabbit Ab to detect GFP but I have encountered a couple of questions:

-Should permeabilization and blocking be apart or together?

-How long should the retina stay in the permeabilization solution (0.1% Triton X-100 in PBS)?

-Is it ok to use a combination of serums as blocking buffer, 3% normal goat serum, 1% bovine serum albumin,and 0.5% Triton X-100 in 0.1 M PBS? What is the rationale behind the combination? Why not just BSA?

-What is a recipe for a primary antibody incubation buffer, I have 1% BSA and 0.1% Triton X-100 in PBS, is this ok? Why not 5% BSA?

-Finally, how long should I leave the tissue with the primary Ab? I've seen protocols doing 3 to 7 days. I don't think I need that long wait as I am looking for superficial GFP.

Thank you for the help.