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> |
Reply-To: | |
Date: | Wed, 17 Mar 1999 16:24:48 +0000 |
Content-Type: | text/plain; charset=iso-8859-1 |
Hello Histonetters, sorry for being somewhat late with my results, it took a while. Aims 2-5µm paraffinsections of rat testis for normal histo and in situ hybridization Problems Sections thinner than 5µm 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 25-35°C 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