RE: block for polyclonals

From:"Tarpley, John"

I didn't respond at first until I re-read your question after Patsy's reply. I'm guessing from what you wrote that your human tissue is formalin fixed rather than the acetone fixation you used for the cell pellets. If so I would definitely retiter the antibody using formalin fixed material. However, another possible problem is that you are adding avidin to your blocking serum. This will tie up any endogenous biotin that may be around, but now you have the problem that you need to neutralize any unbound avidin that may be deposited on your tissue. You can do this by adding biotin to your primary antibody. I find Vector's A/B blocking kit an easy way to do this. You might also try switching to a casine block instead of a serum block. Again, I find Zymed's CAS block to be an easy way to do this. Finally, you are using BSA in your block and perhaps in other buffers. Make sure you are using a high quality BSA. Cheaper, less pure, BSAs can contain proteins that interact with IgGs and lead to nonspecific background. This is especially true for goat primary antibodies. I spend several days trying to titer a goat polyclonal ab and could not get rid of the background stain until I realized that the BSA in my buffer was reacting with the anti-goat secondary. When I changed to a high purity molecular biology grade BSA my problem went away. Similar background problems can be seen with some casine blocks that are not highly purified, but I have not seen the problem with CAS Block. Hope some of these suggestions help.

John E. Tarpley 5-1-A
Associate Scientist
Amgen Inc.
One Amgen Center Drive
Thousand Oaks, CA 91320
These Opinions are my own and not necessarily those of my employer.

-----Original Message-----
From: rueggp []
Sent: Friday, February 22, 2002 2:39 PM
To: Linda Hylander
Subject: 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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