[Histonet] Re: DAB reduction

[David A. Wright]

Hello Naira & Histonet

I'm a bit behind on this thread but you might still be interested in this idea. There is one way you might be able to minimize your DAB signal, but only if it is intensified with a metal salt such as cobalt and/or nickel. You can (slowly) chelate out the metal ions with EDTA. It will still leave the DAB undissolved but now as a golden brown deposit instead of e.g. blue-black. If you determined your end point for the staining reaction with Ni/Co rather than with pure DAB, the residual DAB will be much less intense than you would aim for with pure DAB.

I discovered this by accident, using 0.1% of EDTA dihydrate in buffer, and it took a couple of days to extract at RT. You could try using more or, if your antigen retrieval solution includes EDTA, you could try repeating that (with the heating to speed things up) until the DAB just looks brown.

Since the DAB will still be there, I agree with Amos Brooks that you should re-react with a different chromogen. It is not necessary to switch enzymes, however: peroxidase will still work. The DAB precipitate completely coats the original Ag-Ab-peroxidase, blocking further reaction with the new chromogen (and, if you do more Ag retrieval, you would remove accessible complexes that way). I do sequential peroxidase reactions routinely, with no x-rxn after DAB 1st round.

I hope you were not planning to re-stain for the same antigen; it will be masked by the DAB ppt.

-David
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David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery

ORIGINAL MESSAGES
Re: Histonet Digest, Vol 90, Issue 22  Message: 2
Date: Thu, 19 May 2011 11:55:14 -0500
From: Margaryan, Naira <NMargaryan <@t> childrensmemorial.org> 
Subject: [Histonet] FW: How to remove DAB to restain with DAB

Hello,

I would like to repeat my DAB staining on the same slide with DAB I run before. I know that "acid alcohol" will remove hematoxylin but how to remove DAB?
I appreciate to any suggestion.

Thanks in advance,
Naira

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Re: Histonet Digest, Vol 90, Issue 23 Message: 9
Date: Thu, 19 May 2011 11:23:54 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] FW: How to remove DAB to restain with DAB


DAB reaction is almost permanent and extremely difficult to destain.
René J.

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Re: Histonet Digest, Vol 90, Issue 23 Message: 18
Date: Thu, 19 May 2011 12:26:32 -0700 (PDT)
From: Keith Mikoff <mikoff <@t> pacbell.net>
Subject: [Histonet] Re: Histonet Digest, Vol 90, Issue 22 2. FW: How to remove DAB to restain with DAB (Margaryan, Naira)

Hi Naira,

The short answer is no, it can't be done.

Unless there is a way to release the antibody from the binding site without damaging the binding site, it can't be done. That said, somehow it should be possible to find a reproduce-able fix for this kind of issue, by either mending the binding site, or enhancing the activation of the already applied antibody complex and/or chromogen. Some really cool tricks if practical or possible.


It seems like it should, maybe with the application of an mild acidic or basic wash... with just the right reagent, the right amount, right application and at the right pH. Something to think about.

Keith M. Mikoff, HTL (ASCP) 

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Re: Histonet Digest, Vol 90, Issue 26
Message: 10
Date: Fri, 20 May 2011 16:25:27 -0400
From: Amos Brooks <amosbrooks <@t> gmail.com>
Subject: [Histonet] FW: How to remove DAB to restain with DAB

Hi,
    There is not any practical way to remove the DAB. I think I read about boiling it in a salt (of some sort) solution removing the DAB. Honestly I never bothered trying it as you need to ask yourself "What is this doing to my target antigen?". You should consider just restaining it with the DAB in place using either Alkaline Phosphatase and Fast Red or using a fluorescent detection if the DAB is interfering with the signal.

Amos

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Re: Histonet Digest, Vol 90, Issue 26
Message: 14
Date: Sat, 21 May 2011 09:31:51 -0600
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] FW: How to remove DAB to restain with DAB


I have removed DAB by using the probe cleaning reagents from the Dako Autostainer but Amos is right this destroys the antigen site so it is of no use.  If you really only have that one section I guess you might be able to use it as an H&E.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech

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