slide coating/in situ query

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From:"Marshall, Sharon, Mrs" <marshall@anat.uct.ac.za>
To:histonet@pathology.swmed.edu
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Date:Mon, 20 Sep 1999 16:34:06 SAST-2
Content-Type:text/plain; charset=US-ASCII

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.   
Thanks
Sharon Marshall
e-mail: marshall@anat.uct.ac.za



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