RE: Pretreatment for in situ hybridization

From:"Montague, Donna C"

Christina and interested others:

The point to the use of enzyme treatment of sections for in situ techniques
is not so much antigen retrieval as it is gene or gene product accesibility.

You see, heat induced epitope retrieval is a more accurate term for the
treatment of sections for immunocytochemistry. Heat, ions present in
buffers, and active water molecules used in various itterations in "antigen
retrieval" merely reverse the effects of fixation on the proteins (antigens)
of interest to the immunohistochemistry exercise. 

Enzyme digestion of proteins for in situ techniques is required to remove
endogenous proteins that "cover" genetic material. If sections are
inadequately treated, the probes cannot find the target sequence and false
negatives can occur.

Hope this helps, Donna Montague, M.S.
Research Associate
Physiology/Biophysics and Orthopaedic Surgery
University of Arkansas for Medical Sciences
(501) 603-1239

-----Original Message-----
From: C Sawrenko []
Sent: Thursday, June 06, 2002 10:36 PM
To: HistoNet Server
Subject: Pretreatment for in situ hybridization

Hello Histonetters,
Does anyone out there have any experience using heat (by steam, microwave or
pressure cooker) instead of enzyme (Proteinase K) for pretreatment prior to
in situ hybridization?  We are attempting ISH for kappa, lambda and EBER.
Also has anyone had any success on B5 fixed tissue for the above?
Thanks in advance for your help!

Christina Sawrenko
BC Cancer Agency
Vancouver, BC

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