[Histonet] re: IHC background [Peng] - rat/Ms X-Rxn?
David Anthony Wright
dw18 <@t> uchicago.edu
Mon Dec 29 17:37:54 CST 2003
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!
>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!]
More information about the Histonet