Rene J Buesa wrote: Date: Thu, 27 Sep 2007 08:51:51 -0700 (PDT)
From: Rene J Buesa
Subject: RE: [Histonet] Re: Hydrogen Peroxide quenching
To: "Ford, Judi"
Judi:
Do not be confused at all. This is what happens when working in an unregulated specialty where procedures, like cooking recipes, vary from chef to chef even when all are making "chicken parmigiani". Histology lacks strict methodologies and each one dances to his/her own tune.
People will tell you what they do, and sometimes there is no technical backing for the selection.
Quenching the omnipresent tissue peroxidase is a requirement to prevent backgound noise.
HIER is required to unmask the epitopes and after that step is when I used to quench. Even I did that BEFORE starting the autostainer and by doing so I was able to quench a complete 48 slides run in 5 minutes. cutting 25 minutes from the protocol had this step being included in the automatic run.
My procedure deviates from other procedures as those other deviate from mine. Make you pick but be consistent in how you do it. I hope I have not added more confusion!
René J.
"Ford, Judi" wrote:
Okay, so I'm confused. Some labs do the hydrogen peroxide step before HIER and others afterwards, why? Also, are there differences using hydrogen peroxide diluted in water/methanol/or PBS? Some bring paraffin slides down to absolute alcohol then do the hydrogen peroxide step where others wait until bringing the slides to water before doing the step. Is there some rule of thumb, or do you just figure out which procedure works for that specific antibody in your lab?
I'm still pretty new to this, so sorry if this seems like a dumb question.
Judi F.
Palo Alto, CA
-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Wednesday, September 26, 2007 2:49 PM
To: Daivik_Shah@rush.edu; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Re: Hydrogen Peroxide quenching
You use it immediately after the HIER, before starting the IHC protocol.
René J.
Daivik_Shah@rush.edu wrote:
Hello friends,
I have question regarding Hydrogen peroxide quenching. Is this treatment
applied in Immunohistostain after deparafinization or before application
of secondary antibody? Is that affect any way to stain?
Thanks.
Daivik
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Daivik Shah, Histotechnologist, M.S. Biotechnology
RADC Laboratory, Cohn Building 436
Rush University Medical Center
Tel. 312-563-4891
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