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From:"Connolly, Brett" <> (by way of Marvin Hanna)
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1. We process our tissue for ISH with a routine schedule.

2. All prehybridization solutions should be made up using DEPC-treated water
including the fixative. Don't contaminate slides, containers, glassware with
your hands--always use gloves. Forget about worrying about the xylene and

3. Baskets, glassware and everything else can be rendered RNase-free by
treating with RNaseZap from Ambion, Inc. according to their instructions.

4. If you find a "best" protocol, please let the rest of the world know what
it is!!

Tyramide enhancement (TSA) is great, but may be unnecessary depending upon
the abundance of the specific target mRNA you are seeking.


Brett M. Connolly, Ph.D.
Merck Research Laboratories
Department of Pharmacology
PO Box 4
West Point, PA 19486
Ph. 215-652-2501
FAX 215-652-2075

	From: 	Huib Croes[]
	Sent: 	Monday, January 24, 2000 7:41 PM
	Subject: 	mRNA ISH

	Dear Histonetters,

	We want to perform an mRNA ISH on paraffin embedded mouse brain and
	testis tissues fixed in buffered 4% paraformaldehyde solution.
	So I have a couple of questions:
	1. Does someone has a scheme of how to process these organs (we have
	Shandon Citadel 1000)?
	2. Do we need to do the processing in RNase-free solutions
	water, alcohol) and maybe even in RNase-free xyleen and paraffin ?
	3. How can we make the baskets RNase-free?
	4. What is the best protocol to do an mRNA ISH and what is the best
	enhancement method (tyramide??)
	5. Does anybody has already did this before, if so, please email me

	Thanks in advance

	Huib Croes
	Dept. Celbiology
	Kath. Universiteit Nijmegen
	P.O. Box 9101
	6500 HB Nijmegen

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