From:"Turner, Scott"

I've run into a problem trying to stain both normal and ulcerative colitis
gut by IHC.  I keep getting a strongly positive, highly focal signal that I
know isn't real because it's showing up on both my antibody slides and my
isotype controls even at concentrations as low as 1ug/mL.  This has happened
with different samples of gut tissue, with different antibodies from
different species (both rat and mouse) and with different isotypes (IgG1,
IgG2a, IgG2b).  It isn't a simple mucosal blush--it looks like real signal,
and would be easily misinterpreted without adequate controls.  I don't think
it's endogenous biotin because I'm blocking with sequential avidin/biotin
block and because it's so focal and cellular.   I know it's not the
secondary antibodies or inadequate H2O2 blocking (I'm using Vector Elite
ABC-HRP) because my controls for those are negative.  It's obviously some
kind of interaction between the antibody--any antibody (including isotype
controls)--and some specific cell population, but I cannot figure it out.  I
thought it might be Fc receptors, but I'm blocking with up to 50% normal
rabbit serum and it doesn't make it go away.

I'm wondering if anyone has seen anything similar or might have any
suggestions about how to block this kind of cross-reactivity or nonspecific
interaction or whatever the heck it is.

Scott Turner
DNAX Research, Inc
Palo Alto, CA

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