Re: block for polyclonals

From:Linda Hylander

Thanks to you, Adrian, Gayle, John, Kathleen for your prompt responses. I believe the BSA may be the problem and will add goat + human serum also. Your assistance is greatly appreciated. Histonet is the best.
----- Original Message -----
From: rueggp
To: Linda Hylander
Sent: Saturday, February 23, 2002 11:29 AM
Subject: Re: block for polyclonals

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.

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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