[Histonet] DAB fading/pH range

[Smith, Allen]

Spend $12 on a little bottle of "Permount" (resinous mounting medium)!
HSR is just as good, but harder to find.

Allen A. Smith, Ph.D.
Professor of Anatomy
Barry University
School of Graduate Medical Sciences
            Podiatric Medicine and Surgery
Miami Shores, Florida


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melissa P Wu
Sent: Friday, July 30, 2004 1:06 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] DAB fading/pH range



Hi,

I know this has been addressed in previous posts but I haven't been able to
find
an answer to this question specifically.  What is the pH range that the DAB
stain is stable in?

Supporting info:

I am using DAB (sigma, tablet form) on Drosophila ovaries (enhanced with
Nickel).  After about 8 minutes of the reaction, the tissues will stain
black. 
I mount in 70% glycerol/PBS (pH 7.2), and after a day the stain fades or
disappears. I also tried 90% glycerol/PBS, but the stain faded here as well.

I read somewhere that the nailpolish could affect the staining, so I stored
one
sample in 70% glycerol/PBS, but the color faded in these samples after a
day.

I called Sigma and they suggested using a resin mount, but I don't have
access
to that so I used Vectashield mounting media, the sample's color faded with
this one also.

Also, a side question.  This is my lab's first time using HRP reactions, I
tried
one on a control sample and let it incubate for about 20 minutes in the DAB
reaction.  I was surprised to see the tissue stain black.  I am pretty sure
it
is not reacting to endogenous HRP as I used methanol earlier to destroy the
activity.  I am also pretty sure it is not due to the primary antibody
non-specifically binding, as its antigen is BrdU.  Is it just background
staining that I am seeing, or perhaps my sample was contaminated?

Any help with this situation would be appreciated.


Thanks,

Melissa Wu

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