rat testis paraffinembedding for in situ hybridization

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From:Alexander Brands <alexander.brands@uni-tuebingen.de>
To:HendryC@mar.dfo-mpo.gc.ca, brittman@mail.db.uth.tmc.edu, ausbio@nex.cam.au, grayk@bms.com, joseph.saby@wl.com, mvaughan@sc3102.med.buffalo.edu, uvsgc@msu.oscs.montana.edu, HistoNet Server <HistoNet@Pathology.swmed.edu>
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Date:Wed, 17 Mar 1999 16:24:48 +0000
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Hello Histonetters,

sorry for being somewhat late with my results, it took a while.

Aims
2-5m paraffinsections of rat testis for normal histo and in situ
hybridization

Problems
Sections thinner than 5m were like Parmesan cheese,
Good sections were floating apart in the waterbath.
Differentiating of  toluidine blue stain resulted in total loss of
stain.

Results
Paraffinblocks precooled in the cryostat and sections put in a water
bath of 25-35C gave very nice sections.

0.001% Toluidine blue for 30sec-1min without differentiation or 2 1/2
min "Kernechtrot" (Heaven knows what it is called in english. Chapter )
or Feulgen were good for demonstrating nucleus structures of
seminiferous cells. Only PAS is still not working good, but I did not
try very hard.

In situ hybridization is working well on paraffinsections of the brain.
Signals on the testis are not easily asignable to specific
cellpopulations. I will try a more elaborate perfusion fixation and do
further experiments on cryosections.
The tissues I used at this time were perfused with PBS and
4%PBS-formalin via left ventricle. They were fixed 6-24h in 4%
PBS-formalin or deep frozen. The tunica albuginea of the formalinfixed
testis was cut open prior to fixation . Next time I try cutting off a
small slice. Paraffin embedded tissues were completely dehydrated.

Thanks a bunch for your help

Alexander




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