Re: [Histonet] Hematoxylin staining of frozen samples, where are the nuclei? NUCLEI FOUND!

From:Guillermo Palao

Thanks Gayle for your great advice! I left frozen sections for a few weeks but I recently came back to them and put in practice your suggestion with great results.
  Nuclei now look really nice. I have heard this is a fairly common problem, so I hope more people will benefit from this great tip.
  Best regards,

Gayle Callis  escribió:

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.
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Gayle Callis
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

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