BAckground staining

From:Gayle Callis

You wrote: 

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.

If you suspect Fc recptors, normal serum block may not be enough.  And it
still could be the secondary antibody.  Is secondary antibody adsorbed to
the species you are staining????  And if you use secondary antibodies that
are F(ab')2 fragments of IgG, this helps eliminate Fc receptor problems.
There are also Fc receptor blockers available, but not sure who sells them.  

Just a thought.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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