Re: [Histonet] Re: Hydrogen Peroxide quenching

From:"Joe Nocito"



Judi, Judi Judi (sorry, couldn't help it)

always, always, always. Did I mention always? Always do what is right for=20
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=20
what someone else is using. Sometimes it'll be a trial and error. Whatever 
works best in your situation
    Not every lab is the same and not every tech is the same. Your tissue=20
processing might be different for the next lab, the temp &  humidity in your 
lab may be different from the next lab.  I know this may seem like a cop 
out, but it's true.
    When I was transferred from D.C. to San Antonio to set up an immuno lab, 
I had my former techs FedEx me some reagents to my new lab. Now, I had the 
same reagents, same procedures and same tech. I still had some adjustments 
to do in the new lab because I was getting a high, non-specific background 
staining.
The environment was different as was the tissue processing.
    This is why my philosophy is there is no such thing as a pre-diluted 
antibody. Keep in mind that the companies perform THEIR testing in THEIR 
lab, not yours. I always try a short titer to see if I can get a 1:2 or a=20
1:4 dilution. Look it at it this way, if I can get a 1:4 dilution out of a 
pre-dilute, and the vial has 6 mls, I just purchased 24 mls for the cost of 
6 mls (is that correct? Math was never my subject).
    Did ramble on again? Hey, not that this is a big thing, but I join the 
work force again on Monday. I'm keeping the place secret so I don't get 
called in by CEOs any more.

JTT
----- Original Message ----- 
From: "Ford, Judi" 
To: "Rene J Buesa" ; ; 

Sent: Wednesday, September 26, 2007 5:03 PM
Subject: RE: [Histonet] Re: Hydrogen Peroxide quenching


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
_____________________________
Daivik Shah, Histotechnologist, M.S. Biotechnology
RADC Laboratory, Cohn Building 436
Rush University Medical Center
Tel. 312-563-4891


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