[Histonet] Can DAB block an epitope??

[Amos Brooks]

Hi Gustave,
     I do this frequently, and it works great with some caveats. First it is
best to use a *very* red chromogen (if you choose to use red). I have not
had a lot of luck with AEC the fast red in the DAKO alk phos kit looks great
against DAB.
     Colocalization of antigens can be tricky. In this case
immunofluoresfence really is the way to go. Biocare Medical has some great
Fluorescent secondaries. Colocalized chromogens just look muddy and not very
good. The DAB doesn't really 'block' the epitope, but ut does make it a heck
of a lot harder to see.

Good Luck,
Amos


Message: 19
Date: Fri, 14 Aug 2009 08:37:45 -0700 (PDT)
From: GT Hebert <emerald_lake77 <@t> yahoo.com>
Subject: [Histonet] Can DAB block an epitope??
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID: <661810.77228.qm <@t> web110611.mail.gq1.yahoo.com>
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Hello all,

I was hoping to run a chromogenic immuno to check if two proteins
colocalize.

Since both antibodies are made in rabbit, I was going to stain one and
visualize with DAB and then heat retrieve (pressure or steam) to drop off
the first antibody and restain with my second rabbit antibody using a
different color to visualize.

How likely, if at all will the DAB precipitate block / mask the epitope of
my second antibody if indeed they are in a similar location?

Any info would be greatly appreciated.

Thank you!

Regards.

Gustave

Gustave T. Hebert
Research Scientist I
Metabolic Disease Research
Wyeth Research
200 CambridgePark Drive
Cambridge, MA 02140