[Histonet] Quenching HRP in double IHC

From:Mathew DeGutes

Hi, I am a Research Tech at Northwestern University and I have run into a problem that to me seems
completely bizarre.  I am performing double fluorescent IHC stains using HRP for both stains and
using cy3 and fluorescein to develop on frozen sections.  My problem is this.  After my first
development with cy3 I try to quench the HRP using 6% H2O2 for 10 min.  What then happens is when I 
go to develop my 2nd stain it is being developed by the HRP from the first stain that was
apparently never quenched at all.  I have tested this directly and it continues to be true.  6%
H2O2 should easily knock the HRP out, but it isn't.  All my conventional wisdom is sort of out the
window.  The only thing that I could think would be the cause is water.  Does water destroy H2O2?
Could that inhibit H2O2 action is PBS necessary to preserve it?  Any help is appreciated.

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