slide coating/in situ query

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From:Mike Bromley <>
To:"'histonet'" <>
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 

Mike Bromley
Histology Department
Paterson Institute
Wilmslow Road
M20 9BX
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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