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

[J. A. Kiernan]

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
> > >
> > >
> > >
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