Re: DAB and hematoxylin

From:Amos Brooks

Hi Andi,
    Just a quick dumb question 'bout that ... Wouldn't the Mayer's
hematoxylin disassociate the Ab/Ag complex after the DAB was added too? I am
probably overlooking something and thereby asking a dumb question but the
disassociation of the antibody should occur with or without the DAB when in
the hematoxylin if that was the cause. I don't have any other answer except
to try another hematoxylin and see if it still happens, or to be REALLY
careful to counterstain only after the DAB is on (way to state the obvious
Amos Brooks

----- Original Message -----
From: "Kappeler Andi" 
To: "Histonet" ; "E Sharon Shields"

Sent: Tuesday, April 30, 2002 9:42 AM
Subject: Re: DAB and hematoxylin

Hi Sharon
Hematoxylin has a pH of approximately 2 to 2.5 ... and at this pH all your
antibodies will readily dissociate from their respective antigens, meaning
that all your staining efforts of the last couple of hours are just gone. No
way to save the job, just start over. Sorry to tell you this, but that's the
way it is. Low pH (hematoxylin, acid alcohol, whatever...) - no more bound
antibodies ... and all these low pH reagents will only take seconds to
destroy your previous work! Hope this helps.

Andi Kappeler
Institute of Pathology, University of Bern, Switzerland

-----Ursprüngliche Nachricht-----
Von: E Sharon Shields 
Gesendet: Dienstag, 30. April 2002 15:01
Betreff: DAB and hematoxylin

> Hello Fellow Histonetters,
> I have a question I hope you can answer since it has caused me lots of
> Why will IHC slides not signal with DAB if they where first stained with
Mayers Hematoxylin before the DAB was dropped on? On several occasions, this
morning being the latest, Hematoxylin was mistakenly drop on the slides
before the DAB. Part of  the controls had  DAB dropped on them and the rest
where destained with 70% acid alcohol before the DAB was dropped. None of
the control worked. WHY???  These are controls that  I know are staining
nicely. As I said this is not the first time this has happened.  WE need
> Thanks,
> E.S. Shields
> Baptist Hospital of E TN
> Knoxville, TN

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