[Histonet] DAB fading/pH range

From:Melissa P Wu


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.


Melissa Wu

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