Re: Rat testis paraffinembedding for in situ hyb.

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From:Barry Rittman <brittman@mail.db.uth.tmc.edu> (by way of histonet)
To:histonet <histonet@magicnet.net>
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Alexander,
The symptom you mention would suggest either incomplete dehydration or
infiltration so that there are "dry" areas of tissue in the block.
It could also occur if your water bath is too hot.

Isopropanol is a much more gentle dehydrating agent than ethanol but is
slower and therefore requires longer dehydration times.
Isopropanol is also miscible with paraffin but is slow to be displaced.
Therefore if you are going straight from isopropanol to paraffin you will
need to leave it longer in the wax. If there are traces of isopropanol
remaining in the tissue you may be able to smell this on the cut surface of
the block. If this is the case I would recommend placing block back into
fresh wax.

Fixation, there are several choices. I prefer using Carnoy or Carnoy Lebrun
if the pieces are small.

I am unclear as to why you are differentiating the toluidine blue stain.
You can achieve a very elegant stain using a dilute toluidine blue and
staining progressively.
My personal preference to show the different stages is to use a Feulgen
stain with a delicate light green counterstain.
Barry

At 11:31 AM 2/17/99 +0000, you 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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