I do not currently do PCR or in situ, but if extra cross-linking won't
     cause you any problem, you can try the following procedure for
     promoting adhesion to the slides:

     Coat the slides with 5% gelatin or any other protein-based slide
     adhesive. Cut and mount the sections and allow to dry. Then place the
     slides in a plastic slide box (one of the little 25-slide ones works
     well) with a gauze pad soaked in formalin or formalin-glutaraldehyde.
     Cover and leave overnight at room temp or for an hour at 60C (in a
     fume hood!) Uncover and allow the slides to air out in the hood before
     bringing them into the room.

     This is the strongest tissue-adhesion technique that I know. It has
     not failed me yet with heated mordanting procedures, enzyme digestion,
     strong alkaline stains, etc. I hope this can help.
                                                        Jeff C


______________________________ Reply Separator
_________________________________
Subject: FW: Insitu pcr
Author:  <Patsy.Ruegg@UCHSC.edu> at internet
Date:    11/23/1999 1:58 PM




-----Original Message-----
From: Ruegg Patsy
Sent: Tuesday, November 23, 1999 1:57 PM
To: 'Deborah Moffett'
Subject: RE: Insitu pcr


You can try heating the slides overnight at 60 d C, but this is an common
problem with insitu pcr, the tissue just does not stand up to the  harsh pcr
cycles of heat and chemical enzymes.  It is a real obsticle to doing insitu
pcr.
Patsy Ruegg

-----Original Message-----
From: Deborah Moffett [mailto:DebMoffett@moffett.swinternet.co.uk]
Sent: Tuesday, November 23, 1999 11:41 AM
To: histonet
Subject: Insitu pcr


We are attempting to do insitu PCR on gut and kidney paraffin sections for
bovine adenovirus. We are having some difficulty with disintegration of the
sections during the pcr cycles. Can anyone reccommend a solution to this
problem?
the sections are 4-5um thick , lifted on superfrost plus gold slides and
heated for one hour at 60C.

Deborah Moffett

Veterinary Sciences Division