Re: Rat testis paraffinembedding for in situ hyb.

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From:Mick Rentsch <> (by way of histonet)
To:histonet <>
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Dear Alex,
you may have more than one contributing factor here.
Firstly fixation, it needs to be well fixed and I would suggest Bouins Fluid
for 24hrs.
Secondly the material needs to be well infiltrated with wax which in turn
requires clearing (make sure your processor reagent are not exhausted)
Thirdly, use a larger mold than you might otherwise use so that there is
plenty of supporting paraffin around the edge of the material.
Fourthly, use a lower floatation temperature in your water bath (45C should
be adequate or even less if the problem persists) it may take longer to
properly flatten that's all.
When you do find out what works for you, please let us all know what you
Regards Mike (downunder)
-----Original Message-----
From: Alexander Brands <>
To: HistoNet Server <>
Date: Wednesday, 17 February 1999 9:37
Subject: Rat testis paraffinembedding for in situ hyb.

Has anyone experience with fixation of (rat) testis and
paraffinembedding for subsequent staging of the germcells?

It seems to be impossible to cut the Isopropanol dehydrated tissue
thinner than 5um, which is nessecary for staging the germcells.

The 5um 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 (5um) sections
with 1% HCl-EtOH the color gets lost cmpletely.

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

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