[Histonet] re: IHC background [Peng] - rat/Ms X-Rxn?

From:David Anthony Wright

Hello Baowei Peng (& Histonet)

Amos Brooks has suggested the problem below might be endogenous biotin, with 
helpful suggestions what to do.  I'd like to suggest something else if that 
doesn't work.  A couple of questions-
1) Is your anti-mouse secondary a polyclonal serum (almost certainly) and, if 
so, is it not rat-adsorbed?
2) Is there any reason for there being an inflammatory reaction in your tissue?

There is about a 5-10% overlap in crossreactivity between anti-mouse and anti-
rat IgG antibodies (cf. goat & sheep), depending on how distant the host 
species is.  I've used standard Vector biotinylated anti-mouse IgG secondary 
(BA2000) happily for IHC on rat brain for years with no background but got a 
huge background cross reaction in every animal when staining material 2 days 
after brain surgery when inflammation is extensive.  I know it was X-reaction 
with native rat IgGs in the tissue because the problem vanished when I either 
i) added normal rat serum to the 2ndary incubation mix and so competed out the 
cross reacting moiety or ii) used Vector's rat-adsorbed secondary (BA2001).  
It's more expensive of course!

good luck!
-David Wright
>I'm doing IHC on rat skeletal muscle. I got a heavy background stain on 
>all sections and control section without 1st AB. But there is no stain on 
>the control section without 2nd Ab.
>It looks like the 2nd Ab will react with the rat tissue. My first Ab is 
>derived from Mouse, and second Ab is anti-mouse.
>What could I do to reduce the background stain?-Baowei Peng
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery
Does 2+2=5 for large values of 2?  [YES!]

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