Re: IHC help please - brown everywhere -Reply

From:Tony Henwood <>

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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