Re: Rat testis paraffinembedding for in situ hyb.

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From:Kevin R Gray <grayk@bms.com> (by way of histonet)
To:histonet <histonet@magicnet.net>
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Alexander,

Our lab fixes rat testis in Bouin's fixative.  It is removed in 70%
ethanol containing lithium carbonate during the course of a workday with
the ethanol Li/Carbonate mixture being replaced as the Bouin's comes out
of the tissue.  The testis are then processed overnight under routine
conditions to paraffin.  They are sectioned at 3 um for PAS staining by
our histotechnician.

Kevin Gray
Bristol-Myers Squibb
New Jersey, USA


Alexander Brands wrote:
>
> Has anyone experience with fixation of (rat) testis and
> paraffinembedding for subsequent staging of the germcells?
>
> Problems
> It seems to be impossible to cut the Isopropanol dehydrated tissue
> thinner than 5Ám, which is nessecary for staging the germcells.
>
> The 5Ám sections float apart as soon as they come into the water bassin,
> before I can "catch" them with the slide.
>
> When I try to differrentiate the Toluidinblue stained (5Ám) sections
> with 1% HCl-EtOH the color gets lost cmpletely.
>
> General
> I am trying nonradioactive mRNA in situ hybridization especially on the
> tubuli seminiferi.
> I want to asign the detected mRNA to the different stages of the
> germcells, in particular distinguish between the two compartments made
> by the blood-testis barrier.
>
> Thanks for your help
>
> Alexander Brands, M.D.
> Institute of Toxicology
> University of Tuebingen
> Germany




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