Urea. (Re: [Histonet] Re: Dystrophin antibody)

Tue Apr 20 03:28:51 CDT 2004

Thanks John for giving us this insight into the history of urea. I wasn't
aware of Hausen & Dreyer - and could not tell off hands where I originally
had the urea recipe from, but it must have been Shi et al.. Wish I had my
references a little bit better organized...

Andi Kappeler

----- Original Message ----- 
From: "J. A. Kiernan" <jkiernan <@t> uwo.ca>
To: "Andi Kappeler" <kappeler <@t> patho.unibe.ch>;
<histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, April 19, 2004 5:55 PM
Subject: Urea. (Re: [Histonet] Re: Dystrophin antibody)

> Urea was among the first substances to be used for
> what we now call antigen retrieval, by Hausen & Dreyer
> (1982). They put hydrated paraffin sections into
> 3M aqueous urea (that's 18%w/v) for 5 minutes prior
> to immunofluorescent staining. Their tissues had been
> prepared by freeze-substitution into ethanol, and
> urea treatment would not reactivate antigens after
> aldehyde fixation. Evidently the hydrogen-bonding
> actions of urea could not break the covalent bonds
> formed by formaldehyde or glutaraldehyde, which is
> the presumed mode of action of the hot water used
> in modern antigen retrieval procedures.
>
> Urea has been combined with hot water to enhance real
> antigen retrieval in formaldehyde-fixed tissue.
> Shi et al (1993) microwaved slides in 5% aqueous
> urea and got better results than with lead thiocyanate,
> an earlier retrieval reagent.
>
> (Hausen & Dreyer also commented that longer times than
> 5 min or higher concentrations of urea caused detachment
> of sections from slides.)
>
> Refs:
>   Hausen P., Dreyer, C. 1982. Urea reactivates antigens
> in paraffin sections. Stain Technol. 57: 321-324.
>   Shi, S.R. et 4 al. 1993. Antigen retrieval using citrate
> buffer or urea solution for immunohistochemical demonstration
> of androgen receptor in formalin-fixed paraffin sections.
> J. Histochem. Cytochem. 41: 1599-1604.
> -- 
> -------------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
>    kiernan[AT]uwo.ca
>    http://publish.uwo.ca/~jkiernan/
>    http://instruct.uwo.ca/anatomy/530/index.htm
> _______________________________
> Andi Kappeler wrote:
> >
> > 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
> > > >
> > > >
> > > >
> > > > _______________________________________________
> > > > Histonet mailing list
> > > > Histonet <@t> lists.utsouthwestern.edu
> > > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> > > >
> > >
> > >
> > > _______________________________________________
> > > Histonet mailing list
> > > Histonet <@t> lists.utsouthwestern.edu
> > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> > >
> > >
> > >
> >
> > _______________________________________________
> > Histonet mailing list
> > Histonet <@t> lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>