Re: DAB and hematoxylin

From:Kappeler Andi

Hi Dana
H&E stained slides can work very well for IHC, the reason why you don't
encounter the 'Ab-Ag dissociation problem' is the following: Before you do
your IHC on H&E slides you remove the coverslip, you go into some buffer
with your slide - and there you are at pH 7.5 again, where Ab-Ag
interactions will work as smoothly as on a freshly cut slide (that you will
stain with neutrally buffered reagents as well). As I mentioned before, it's
NOT the hematoxylin as a reagent, it's the low pH of the hematoxylin
staining solution! You can dip your slides in any buffer of pH 2.0 - 2.5
before applying your chromogen and you will have the same effect as if you
put it in hematoxylin. Again, we are talking of the hematoxylin staining
solution (pH 2-2.5), not of the hematoxylin that is deposited on a stained
slide, which, at this point, will exert virtually no influence on the pH of
reagents that you apply, in  particular as these are usually buffered around
Institute of Pathology, University of Bern, Switzerland

-----Ursprüngliche Nachricht-----
Von: Dana Settembre 
An: ; ;

Gesendet: Mittwoch, 1. Mai 2002 19:49
Betreff: Re: DAB and hematoxylin

> Mr. Amos Brooks has a good point.  AND what about the fact that I am
> often given an H&E slide to use for an immuno stain?  They have always
> I must admit that I have not run all antibodies on H&E slides.  But I
maybe a different Hematoxylin would be the way to go.
> Dana Settembre
> University Hospital-UMDNJ
> Newark, NJ
> >>> Amos Brooks  04/30/02 04:36PM >>>
> 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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