Joe,

I must respectfully differ with your advice:

>>always, always, always. Did I mention always? Always do what is right
for 
the lab you are in. I have tried H2O2 blocking before HIER, after HIER, 
after the secondary antibody and everywhere in between. As long as you
block 
before the DAB you should be fine. Most of the time you can get away
with 
what someone else is using. Sometimes it'll be a trial and error.
Whatever 
works best in your situation<<

If you block use H2O2 after the secondary antibody, you will quench the
the HRP. I think you meant to say BEFORE the secondary antibody, not
just anytime before the DAB.

Some folks report needing to do the H2O2 quenching after HIER as it has
been demonstrated to reactivate HRP if done prior to HIER. That has not
been my experience, but because of this possibility, I do the quenching
after HIER and before the protein block.

Some folks are worried the H2O2 will negatively affect the binding of
the antibody to their target, and therefore will not do the block until
after the primary antibody incubation. If that makes them sleep better
at night, then go for it. I don't think that aqueous H2O2 has as much an
effect on immunoreactivity as solution using methanol.  There are
published reports where CD markers were negatively affected in
cryosection using H2O2 in methanol as a block prior to primary antibody
incubation. It may matter less in paraffin embedded material, but why
take the risk? We use aqueous H2O2 and do not make it in PBS, only in
distilled or DI water.

Hope this helps!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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