[Histonet] Re: Dystrophin antibody

[Andi Kappeler]

Hi Steven

Urea is a denaturing reagent which found its way into IHC-labs probably from
molecular biology. There it was used in DNA sequencing gels, before the
arrival of the automated capillyry sequencing machines used today. Its
'task' was to help keep denatured DNA in the single strand conformation.
I've seen the recipe for this 'buffer' (it's not really a buffer, because at
pH 9.5 the buffering capacity of tris is close to zero) published a couple
of years ago (would have to unearth the reference ...) - we tried it, and it
worked quite well, at least for some antibodies. What it actually DOES on
our tissue sections is beyond my knowledge, however with many of all these
retrieval recipes we do probably not exactly know, what all the ingredients
in buffers, etc. do. As with other high pH retrieval solutions,
urea-containing buffer also reveals endogenous biotin, so you have to do an
avidin-biotin block at least for such citical tissue samples as kidney,
liver, etc. or use a non-biotin dependent visualization system. Hope this
helps.
Andi

----- Original Message ----- 
From: <steven.p.postl <@t> abbott.com>
To: "Andi Kappeler" <kappeler <@t> patho.unibe.ch>
Sent: Wednesday, April 14, 2004 9:04 PM
Subject: Re: [Histonet] Re: Dystrophin antibody


> Andi,
>
> If I may ask, what is Urea used for?
>
>
>
>
> "Andi Kappeler" <kappeler <@t> patho.unibe.ch>
> Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
> 04/14/2004 11:33 AM
>
>
>         To:     "Histonet" <HistoNet <@t> pathology.swmed.edu>
>         cc:
>         Subject:        [Histonet] Re: Dystrophin antibody
>
>
> Hi Cheryl
> we use clone 13H6 (Novocastra, NCL-DYSA) at 1:50 after HIER in 100 mM Tris
> -
> 5% Urea, pH 9.5. Other high pH retrieval buffers may work as well. Hope
> this
> helps.
>
> Andi Kappeler
> Institute of Pathology, University of Bern, Switzerland
>
> ----- Original Message ----- 
> From: "Cheryl Crowder" <ccrowder <@t> mail.vetmed.lsu.edu>
> To: "Histonet" <histonet <@t> pathology.swmed.edu>
> Sent: Wednesday, April 14, 2004 5:58 PM
> Subject: [Histonet] Dystrophin antibody
>
>
> > Hi - I have a researcher who would like to do Dystrophin IHC on
> > formalin-fixed, paraffin embedded tissue.  Does anyone know of an
> antibody
> > which will work.  Thank you, Cheryl
> >
> >
> > Cheryl Crowder, BA, HTL(ASCP)
> > Chief Technologist
> > Anatomic Pathology
> > Department of Pathobiological Sciences
> > School of Veterinary Medicine
> > Louisiana State University
> > Skip Bertman Drive
> > Baton Rouge, LA  70803
> >
> > 225-578-9734
> > FAX:  225-578-9720
> >
> >
> >
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>
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