Re: DAB and hematoxylin

From:nina leek

It's my understanding that the DAB oxidation product is precipitated into the
tissue, and probably even chemically bonded to it.  So, it doesn't matter if
the Ab/Ag complex is dissociated at that point, the stain from DAB will stay
Adrian Leek.

Amos Brooks wrote:

> 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
> right).
> 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 
> An: 
> 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
> grief.
> > 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
> help!!!!!
> > Thanks,
> > E.S. Shields
> > Baptist Hospital of E TN
> > Knoxville, TN
> >
> >

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