Try blocking the sections with serum from the species the antibody was raised
in, and preincubating the antibodies (we use 30 min at room temp.) with serum
of the species you are staining (ie rat). Do you incubate at room temperature
or at 4oC? You can replace a 1 hr incubation at RT with an overnight
incubation at 4oC. I find that this often gives denser and more specific
staining. In two of the protocols I use, I lose much of the immunoreactivity if
I fix with paraformaldehyde. Instead, the tissue is frozen unfixed, sectioned,
and the sections fixed in acetone (2-5 min) and dried with cool air.
