Re: block for polyclonals
You might have to use a different antibody titer for frozen sections than
you do for cell pellets. It will help to put some normal serum from
the animal you are staining in your blocking reagent. If you are
staining human tissue put 10% human serum in with your goat serum.
Another possible fix would be to use an avidin/biotin block or a detection
system that doesn't use AB such as DAKO Envision two step labelled polymer
system, in case your background is endogenous biotin which may show up
in your frozen sections and not in your cell pellets.
Patsy Ruegg
Linda Hylander wrote:
I
was wondering if anyone has suggestions to eliminate background when using
polyclonal Ab. I work with frozen cell lines and tissues, I rarely do formalin
fixed IHC.Problem:
I titered out a primary polyclonal Ab fine with no background on a frozen
cell pellet, acetone fixed slides, Ab concentration 2.5ug/mL. I stained
frozen tumor breast tissues, had loads of background on my isotype control
sections. My secondary Ab is goat anti-rabbit, biotinylated, I blocked
with 10% goat serum, avidin and BSA. I appreciate any ideas and many thanks.
Linda Hylander HT, IHC(ASCP)
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