[Histonet] How to Permeabilize thick cultures?

[Pixley, Sarah (pixleysk)]

Dear List:
I have some thick explant-like cultures (many cell layers thick). We are
having great difficulty getting antibodies to penetrate. All the staining
occurs in thinned out edge areas but nothing stains in the central regions,
where I know there are cells that should be positive for this antigen. We
have tried: 1) using 1% Triton in the blocking and primary antibody steps
and letting the antibody incubate for 2 days, while secondary and ABC
reagents were left on for 4-6 hours. 2) we have permeabilized by placing the
cultures (grown on filter papers) in acetone (chilled with dry ice/ethanol)
for 5 mins. This gave a hint of increased staining, but not as good as for
monolayer cultures. Today we are trying cultures treated with acetone for 15
minutes. Antibodies to antigens on or very close to the surface work well,
but anything that needs to penetrate is not working.

Does anyone have any other ideas? Should I increase staining to a week? 
Thanks in advance.
Sarah Pixley
Univ. of Cincinnati