DAB fading

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From:"Rebecca Hartley" <rebeccahartley@earthlink.net>
To:"HistoNet Server" <HistoNet@Pathology.swmed.edu>
Date:Tue, 29 Jun 1999 16:17:19 -0700
Content-Type:text/plain; charset="iso-8859-1"

I used Sigma's metal-enhanced DAB tablets on 40 um free-floating sections
(after primary antibody and ABC elite kit), followed by a counterstain with
Mayer's Hematoxylin, rinses in ddH20 and aqueous mounting in Biomeda's
Gel/Mount for aqueous and dry mounting.

Here's the problem: Immediately after the DAB step, the staining looked very
strong.  However, after the hematoxylin step the intensity of staining had
decreased, and one day after mounting the sections were light brown instead
of the rich black color they had been right after the DAB procedure.  It was
my understanding that the DAB precipitate was insoluble --  ?  Should I be
using a different hematoxylin or different counterstain altogether (I don't
mind the black stain/blue nuclei)?  Could this be a problem with
metal-enhanced DAB (and not plain DAB), or with the mounting media?  I am
not working in a histochem lab, so do not have access to a slide heater etc
for gelatin-coated slides for dehydration, if this is the problem.  Is there
another way to mount and dehydrate the tissue?  Does air drying the tissue,
with no EtOH or Xylene steps work?

The tissue I am trying to stain is very precious and limited, so I can't
experiment with different permutations.  I would REALLY appreciate any help
or suggestions that anyone could give me.


Rebecca Hartley, Ph.D.
Layton BioSciences, Inc.
Gilroy, CA


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