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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