Re: [Histonet] Hematoxylin staining of frozen samples, where are the nuclei? NUCLEI FOUND!
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.
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
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
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:
> 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.
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Veterinary Molecular Biology
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406 994-4303 (FAX)
Guillermo Palao, MD. Ph.D.
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Centro de Investigación
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