[Histonet] Endogenous Peroxide staining help

[Lewis, Patrick]

Hi everyone,

I seem to have a lot of endogenous Peroxide background staining in my FFPE IHC.  Human tonsil tissues, with some attached muscle.

I do a H202 block at 0.3% H202 in TBS pH 8.0 for 30 minutes.

Then I wash x3  with TBST 0.05% Tween20, pH 8.0

Then I serum block with 2% NGS in TBST for 30 minutes.

Then I wash x3 TBST

Then I add my primary antibody in 2% NGS in TBST overnight.

Then I wash x3 TBST

Then I add my HRP-labeled 2ndary antibody for 30 minutes.

Then I wash x3 TBST

Then I have add my AEC Substrate.

Should I increase the concentration of H202?
Should I increase the time in H202?

Is there a different step in the IHC protocol that is better for blocking endogenous H202 activity?
Is it possible to lose/reverse the blocking for some reason?

I am concerned that my HRP labeled secondary may be to blame.

Thoughts?

Patrick.

Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115

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