----- Original Message -----

From: rueggp

To: Linda Hylander

Cc: histonet@pathology.swmed.edu

Sent: Saturday, February 23, 2002 11:29 AM

Subject: Re: block for polyclonals

Linda,
John's reply reminded me that BSA can surely be your problem.  I stopped using BSA and do much better with serum block both from the host of the secondary and serum from the species being stained.  This would be like having a human absorbed secondary.  I use this serum block before the primary antibody and then again before the secondary.
Patsy

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)