false positive with HRP and DAB


I apologize for hitting send before adding subject on last post!

Good morning Histonetters!
Negative controls (primary ab omitted) for my HRP-conjugated IHC in ffpe
tissue show staining with DAB even though I am doing block for endogenous
peroxidase (5 min in 3% H2O2)after rehydration, before blocking with serum.

I have seen in the archives that one may use a lower concentration of H202
(diluted in PBS)and incubating longer. Is this worth trying?

I will be replacing my bottle of peroxide, then try extending the
incubation time in peroxide, but would like some expert advice on optimal

I am using the Vectastain ready-to-use universal biotinylated secondary ab,
should I consider trying to dilute it?
Many thanks.
Maureen Loomer
MS candidate (Cell Biology)
East Carolina University 

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