Re: DAB and hematoxylin

From:Kappeler Andi

Hi Amos
You are absolutely right, when you dip your slides in hematoxylin after
having applied DAB, your Ab/Ag complexes will dissociate - but then, who
cares at this point of the protocol (as Adrian stated): the (brown) color is
deposited on your tissue, there is no longer a need for primary, secondary
antibodies, enzymes and the like. Two more words: hematoxylin is not the
culprit as hematoxylin, it's the low pH. We are not using Mayer's, but
Gill's (pH 2.4), and it's the same (it happens once every two-three years
that a batch of 200 slides goes into hematoxylin before it has been in the
chromogen/substrate solution - no need to mention that these are not exactly
very joyful events ...). Second: DAB, when precipitated, is very dense and
may infact 'trap' your antibodies on the tissue, in particular when you are
demonstrating a very abundant antigen. This is one of the reasons why you
may face problems when doing simple, straight-forward double staining with
two primaries from the same species: your first primariy antibody may have
it's feet (i.e. antigen binding site) 'caught' in DAB and the Fc portion may
be exposed above the DAB, where your second visualization system may
interfere with it. Old double staining protocols suggested a 'stripping'
step after the first visualization (with DAB), which was an immersion into
glycin buffer of pH 2.2. This had the obvious goal of removing all the
antibodies from your first staining round, before starting over with a new
primary antibody (of the same species) and the second visualization. It
works reasonably well with some primaries, but it may not work that well
when your first antigen is very abundant and you have a lot of DAB that
keeps at least some of the reagents from the first staining on the tissue,
in spite of the low pH step. Wow, is this complicated, wish I could quickly
explain this with a paper and a pencil ... Nevertheless hope it was +/-
Best regards

-----Ursprüngliche Nachricht-----
Von: Amos Brooks 
An: Kappeler Andi ; Histonet

Gesendet: Dienstag, 30. April 2002 22:36
Betreff: Re: DAB and hematoxylin

> 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
> probably overlooking something and thereby asking a dumb question but the
> disassociation of the antibody should occur with or without the DAB when
> the hematoxylin if that was the cause. I don't have any other answer
> 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.
> way to save the job, just start over. Sorry to tell you this, but that's
> 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,
> 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
> 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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