[Histonet] Re: Temperatures for IHC

[John Auld]

Ronald
Be careful using higher temperatures. 2 - 3 years ago I did a study using 6
Abs at different temperatures as well as asking on Histonet. Some Abs, ER &
PR particularly, give weaker staining as well as staining fewer cells at
temperatures above 25 C. If you want to stain at a higher temp I suggest
validating every Ab before hand.

Cheers

John

John Auld MSc CSci FIBMS
BMS 4
Dept of Histopathology and Clinical Cytology
Arrowe Park Hospital
Arrowe Park Road
Upton
Wirral

Internal extn 2560
External Tel 0151 604 7025

Message: 7
Date: Wed, 1 Sep 2004 14:24:34 -0400
From: "Ronald P. Wilson" <rpw4 <@t> psu.edu>
Subject: [Histonet] Temperatures for IHC
To: "Histonet" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <FEEHJCAMKILNFDNCLDJPEEAIDLAA.rpw4 <@t> psu.edu>
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We are in the process of optimizing our IHC procedures. We have switched to
using some of the reagents from Phoenix Biotechnologies. Their recommended
protocols suggest primary and secondary antibody as well as strepavidin and
chromagen incubations be done at 55ºC for much shorter time periods (4-10
minutes). I have also heard that some labs do their incubations at 37ºC. Is
it common practice to use temperatures higher than room temperature? Are
there advantages other than just shortening the incubation time? If anyone
is using the higher temperature, how do you do this for bench-top
procedures? I know that some of the staining stations such as Microprobe
have built-in incubation chambers to do this.

Ronald P. Wilson, V.M.D., M.S.
Associate Professor
Department of Comparative Medicine, HO54
Penn State University College of Medicine
M. S. Hershey Medical Center
500 University Drive
Hershey, PA 17033
phone:  717-531-8460
fax:       717-531-5001
e-mail:  rpw4 <@t> psu.edu