[Histonet] Sections falling off during in situ

From:Rebecca Jo

Hello all-

I'm doing an in situ hybridization with adult frog eye sections.  I cut the
sections on a cryostat about 20 microns thick.  I've been having problems
with my sections falling off after treatment with proteinase K.  My protocol
calls for 10ug/ml of proteinase K; however, I have played around with
different concentrations, even going down to 1ug/ml.  It seems no matter what
I do, my sections tend to fall off.  Please, what am I doing wrong?

Also if it helps, before treatment with proteinase K, I dry the sections at
50C for 10 minutes then and treat with 4% PFA 20 minutes.

Thanks all.

Rebecca E. Jo
UNC-School of Medicine
Department of Cell and Developmental Biology
CB# 7090

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