[Histonet] IHC on frozens

[Sarka Lhotak]

Hello Netters,

I am trying to get some antibodies work on mouse tissue (see my
previous posting). Since I have not been successful on paraffin
sections I now turned to frozens where I have no experience. I am
getting the same staining (or background of course) on positive and
negative slides (antibody omitted). Even antibodies that I know work
very well on paraffin (my positive control), give me no specific
staining and a background mainly on connective tissue. I've searched
for a proper fixing, drying, dipping in water etc. procedures prior to
staining. I found it very confusing, people swear by different, even
opposing protocols.
So I have a few questions:
What is the best way to prepare/store your sections prior to staining?
Is quenching peroxidase and blocking the same as on paraffin?
Generally, are the primary and secondary antibody concentrations the
same as on paraffin?
Is the MOM problem worse on frozens than on paraffin?

All suggestions are welcome.

Desperately,

Sarka Lhotak

McMaster University
Hamilton, Ontario