Dear Sharon,

> 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.   

The APTES technique should not be this difficult. I have used the 
APTS slides described on my web page for Heat antigen retrieval as 
well as in situ hybridisation for mRNA and DNA and have not noticed 
any problems.

Regards.... Tony
.
Tony Henwood
Senior Scientist
Anatomical Pathology
Royal Prince Alfred Hospital
Sydney, AUSTRALIA

http://www2.one.net.au/~henwood
http://www.pathsearch.com/homepages/TonyHenwood/default.html