Re: IHC help please - brown everywhere -Reply
From: | Tony Henwood <AnthonyH@chw.edu.au> |
Greg,
Why do the blocking after the localising antibody step? Isn't it possible
that the localising antibody might bind non-specifically to the tissue? It
seems that it would be better to do it after the endog peroxidase block
before the localising antibody incubation.
Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318
>Second, you appear to be missing two other key blocking steps.
One is a normal serum block. This is a step that can be inserted
either before or after the primary antibody, and is usually a normal
serum blocking solution made of the same species as your
secondary Ab (e.g. if using Goat anti-mouse HRP, use a 1%
solution of normal goat serum) to block nonspecific binding sites in
the tissue. The other missing blocking step, is a step to block
endogenous biotin in the tissue. In mammals at least, the gut can
be rich in biotin. So the Avidin label may well be sticking to
caterpillar biotin rather than your reagent biotin. Someone else on
this list can give you a homemade recipe for biotin block. I just buy
a biotin blocking kit from Dako.
Cheers! Greg
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