Re: DAB and hematoxylin
That makes sense. As I said, I probably overlooked something. To put a
positive spin on this, if you accidentally put the wrong antibody on the
slide, you could fix it by an acid rinse before the DAB is put on then after
rinsing it you could start over with the right antibody. You shouldn't have
any worries about the original Ab/Ag interfering with the second. That could
be a good thing to know (Thank you Sharon) if you forget to get your coffee
in the morning.
----- Original Message -----
From: "nina leek"
To: "Amos Brooks" ; "Histonet"
Sent: Tuesday, April 30, 2002 6:05 PM
Subject: Re: DAB and hematoxylin
> It's my understanding that the DAB oxidation product is precipitated into
> tissue, and probably even chemically bonded to it. So, it doesn't matter
> the Ab/Ag complex is dissociated at that point, the stain from DAB will
> 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?
> > probably overlooking something and thereby asking a dumb question but
> > 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
> > 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
> > antibodies will readily dissociate from their respective antigens,
> > that all your staining efforts of the last couple of hours are just
> > 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
> > 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
> > 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
> > 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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