Guillemo,

Hooray for success.  This is a technic I learned from Dr. Chris van der 
Loos, he needs a round of applause,  I just passed on his solution to the 
problem.

Gayle Callis

I think what is most important is that At 01:24 PM 5/8/2006, you wrote:
>Thanks Gayle for your great advice! I left frozen sections for a few weeks=20
>but I recently came back to them and put in practice your suggestion with=20
>great results.
>
>Nuclei now look really nice. I have heard this is a fairly common problem,=20
>so I hope more people will benefit from this great tip.
>
>Best regards,
>
>Guillermo
>
>Gayle Callis  escribió:
>Guillermo
>
>It is because they are minimally fixed with acetone, correct? The nuclei
>are not lost, just look funny like bubble with blue rim or vacuoles with
>blue rims. I know the problem. A lot of the problem is caused by the kind
>of water you rinse with to stop the chromogen reaction - distilled water is
>not ideal.
>
>Chris van der Loos has the answer for this by post fixing an IHC stained
>frozen section with formalin before doing the hematoxylin staining. For
>further discussion, you can contact him at "C.M. vander Loos"
>. This was discussed in the past year at great
>length by him and others on Histonet. This post fixation with formalin
>seems to restore and fix the nuclei - it is important to remember, acetone
>is a precipitating fixative and this is very minimal as compared to fixing
>a frozen section with NBF or paraformaldehyde, cross linking fixatives.
>
>How he does this:
>
>Do all your IHC staining up through completion of chromogen step, and after
>final rinse (do not use distilled water, tap water or even PBS may be
>better) then:
>
>Postfix in neutral buffered formalin for 3 minutes
>Rinse 2 min with gently running tap water,
>Brief rinse in distilled water
>Do the Hematoxylin counterstain
>
>See if this helps you
>
>
>
>
>
>At 03:22 PM 4/4/2006, you wrote:
> >Dear histonetters,
> >
> > When I stain samples placed on VWR frosted slides with Gills
> > hematoxylin, nuclei are nicely stained. However, when the same samples
> > are counterstained after passing through all necessary steps involved in
> > immunohistochemistry, hematoxylin staining of nuclei is simply horrible.
> > Is it possible that nuclei are lost in the different washing steps?
> > Please put forward any suggestions you might have.
> >
> >
> >
> >
> >---------------------------------
> >
> >LLama Gratis a cualquier PC del Mundo.
> >Llamadas a fijos y móviles desde 1 céntimo por minuto.
> >http://es.voice.yahoo.com
> >_______________________________________________
> >Histonet mailing list
> >Histonet@lists.utsouthwestern.edu
> >http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>Gayle Callis
>MT,HT,HTL(ASCP)
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367
>406 994-4303 (FAX)
>
>
>
>
>
>Guillermo Palao, MD. Ph.D.
>Laboratorio de Reumatología
>Centro de Investigación
>Hospital 12 de Octubre
>Avda de Córdoba s/n
>Madrid 28041
>Spain
>
>
>
>LLama Gratis a cualquier PC del Mundo.
>Llamadas a fijos y móviles desde 1 céntimo por minuto.
>http://es.voice.yahoo.com 
>


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