slide coating/in situ query
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From: | Mike Bromley <MBromley@PICR.man.ac.uk> |
To: | "'histonet'" <histonet@pathology.swmed.edu> |
Reply-To: | |
Date: | Tue, 21 Sep 1999 09:08:52 +0100 |
Content-Type: | text/plain; charset="us-ascii" |
Hi Sharon
I have found the best way to coat slides for in situ is briefly:
Use clean slides straight from the manufacturer
Wear gloves to put into racks
Bake at 200o for 4 hours to destroy RNases
When cool dip as follows:
Soak in acetone for 5 minutes (this removes traces of grease)
Then 2% APES in acetone for 5 minutes (this longer time makes the
sections stick better)
drain but don't dry
rinse in DEPC treated water 10 complete dips and drain
then dry in a fan oven at 45o
I find if you heat the slides too much after the APES treatment they
lose their stickiness, especially at temperatures designed to destroy
RNases
Mike Bromley
Histology Department
Paterson Institute
Wilmslow Road
Manchester
M20 9BX
UK
Date: 20 Sep 1999 11:11:01 -0500
From: "Marshall, Sharon, Mrs"
Subject: slide coating/in situ query
Dear histonetters,
Would like some advice on the following. We wish to coat some slides
with APTES before doing in situ hybridisation. I would prefer to use
extran as an initial wash as opposed to chromic acid as I feel more
comfortable working with a detergent than an acid. I am using the
extran at a dilution of 10%. Is this too high or could I get away with
a lower dilution? Apparently the acid would destroy any protein
present. Would the detergent do this as well? I plan to leave the
slides in extran overnight. The method I have also recommends a wash
after the extran step at 60 degrees for 2hrs in tap water. Could
anybody explain or has a gremlin crept in when the method was passed
along. I presume that washing in tap water for 2hrs at R.T. would be
just as sufficient? The next step is a drying procedure at 160
degrees for 2hrs. I guess any drying method is suitable? Once the
slides haved cooled we will then coat them, (2% Aptes in
acetone)5secs., rinse them in acetone and sterile distilled water and
dry them. We also then DEPC treat and bake the slides before using
the slides for in situ.
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