[Histonet] Re: Can PBS cause background

[Brian Chelack]

Hello Carolyn;

In the late 80's I did use an alternate procedure to
inactivate endogenous peroxidases that included sodium
borohydride. We were testing methods for antigens that were
sensitive to H2O2-methanol. My memory is dim, but as I
recall the procedure involved the immersion of the slides in
0.1% phenylhydrazine at 37C for 1 hour followed by immersion
in 0.5 mg/ml NaBH4 for 10 minutes. In my experience bubbles
under the sections were not a problem, but the treatment was
tough on the sections. Phenylhydrazine is also not a very
nice chemical to work with. If you are looking for a
non-methonal method try using PBS + 2% H2O2 + 0.1% Na Azide.

As far as PBS vs Tris based buffers is concerned, I believe
the amino groups in the Tris can act to reduce nonspecific
antibody binding. Over the years I have used both Tris and
PBS based buffers and have seen differences in the staining
intensity that is antibody specific. That is, certain
antibodies do work slightly better with one buffer or the
other. Ultimately the differences were not significant
enough to warrant the use of one buffer over the other. We
use PBS but Tris will work just fine.

Brian Chelack
Prairie Diagnostic Services