Judy (and anyone else interested!) -

I still don't know why this problem with DAB loss with aqueous mounting is 
happening.  I had done this in the past with no problem, so ???  The only 
real difference from then to now was the DAB itself - this was the first 
time I've used the Sigma DAB tablets. But, I tried air drying the tissue 
and mounting with Cytoseal 60 (Stephens Scientific) and everything looks 
just fine.  I didn't use any alcohols, so you may be able to do this also. 

If anyone has any ideas on what's going on with this, I'd love to hear 
them!

Rebecca Hartley
Layton BioScience, Inc.
Gilroy, CA
rebeccahartley@earthlink.net


Re:
Date: 21 Jul 1999 09:25:44 -0500
From: Judy Trogadis <judy@playfair.utoronto.ca>
Subject: DAB question

Dear Histonetters,

We are staining cryosections of mouse embryos which have been frozen,
sectioned and then fixed in paraformaldehyde. Immunostaining was carried
out with the Vector Elite ABC system and the chromogen is DAB. After
the DAB reaction is terminated with water, we don't dehydrate because
we are also looking at retina which is easily damaged by alcohol,
Therefore,we just use an aqueous mounting medium, either Ferrant's medium, 
or Aquamount.

After coverslipping, the retinas look great. The mouse embryo DAB staining, 
however, quickly fades until it is barely visible, in a couple of hours.
Any theories of how this could be happening?

I should add that the retinas are fixed after removal from the animal, 
prior
to freezing, but how could that make a difference to disolving the DAB
precipitate?