Thank you guys,
Thank you very much for your reply. I will try treatment of quenching at
different time and find the correct time.
Daivik
_________________________=5F______________
Daivik Shah, Histotechnologist, M.S. Biotechnology
RADC Laboratory, Cohn Building 436
Rush University Medical Center
Tel. 312-563-4891
This message and any attachments contain information for the exclusive use
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"Joe Nocito"
09/26/2007 06:21 PM
To
"Ford, Judi" , "Rene J Buesa" ,
,
cc
Subject
Re: [Histonet] Re: Hydrogen Peroxide quenching
Judi, Judi Judi (sorry, couldn't help it)
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
Not every lab is the same and not every tech is the same. Your tissue
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
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
_________________________=5F___
Daivik Shah, Histotechnologist, M.S. Biotechnology
RADC Laboratory, Cohn Building 436
Rush University Medical Center
Tel. 312-563-4891
This message and any attachments contain information for the exclusive use
of the individual or entity to whom it is addressed and may contain
information that is privileged, confidential and/or exempt from disclosure
under applicable law. If the reader of this message is not the intended
recipient, or the employee or agent, you are hereby notified that any
distribution or copying of this communication is strictly prohibited. If
you received this message in error, please phone me immediately at
312-563-4891.
_________________________=5F_____________________
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