The NSH Manual needs to be seriously updated. There is no protocol for mouse brain that could help this guy with his problem.
Andi On Feb 22, 2011, at 2:02 AM, Margaret Blount wrote: > I agree with all the comments so far, and I think all the steps are far > too long, particularly the overnight in molten wax. You are effectively > cooking your samples. I would avoid overnight in 100% ethanol too; it > will tend to harden the tissue. > Get hold of the manual for animal tissues available from the Society for > histotechnology. I found this invaluable and now most of my tissues can > be sectioned without soaking at all. I just chill them slightly, if > necessary. > > Good luck with your samples. > > Margaret > > Miss Margaret Blount > Histology Manager > Metabolic Research Laboratories > Level 4 Institute of Metabolic Science > Box 289, Addenbrooke's Hospital > Hills Road, Cambridge, CB2 0QQ > > Tel 01223 769061/336079 > > > -----Original Message----- > From: [email protected] > [mailto:[email protected]] On Behalf Of Noel > Gray > Sent: 21 February 2011 20:55 > To: [email protected] > Subject: [Histonet] Formalin-fixed paraffin embedded question > > I am having an issue with formalin-fixed, paraffin embedded tissue that > I am > sectioning. I am using a microtome to cut the tissue (mouse brain, > cerebellum and spinal cord) in 10 um sections which I will stain using > cressyl violet. Sometimes, the tissue in a block will splinter once it > hits > the blade. Usually all samples from the same animal splinter but this is > not > always the case. If I put the block into the water bath (sectioning > surface > exposed to water or not) this seems to stop the splintering for 1-200 um > worth of tissue. However, I am afraid this may bring error into the > histological analysis of my tissue. > > I assume it has something to do with the protocol I am using to prepare > the > tissue. I guessed that maybe the dehydration, alcohol clearing, or > paraffin > infiltration are not complete, resulting in the problem I have. However, > I > looked at various FFPE protocols and each of my wash steps are longer, > which > may be the problem? I was wondering if anyone has encountered this > before, > or if anyone knows exactly what is going on with my tissue and how I can > fix > it? Thank you. > > Here is my protocol: > > -Anesthetize mouse followed by a system flush of 30 ml of PBS and then > slow > perfusion of 50 ml of 4% PFA. > -Brain and spinal cord are removed as a single, in tact unit and placed > into 70% ethanol for 4 hours > -80% EtOH for 4 hours > -90% EtOH over night > -100% EtOH #1 for 4 hours > -100% EtOH #2 for 4 hours > -100% EtOH #3 over night, forebrain cerebellum and spinal cord are > separated > -Xylene wash #1 for 4 hours > -Xylene wash #2 for 4 hours > -Xylene wash #3 over night > -Moulton paraffin wash #1 for 4 hours > -Moulton paraffin wash #2 for 4 hours > -Moulton paraffin wash #3 over night > -Tissue is embedded and then sectioned into 10 um sections > > Again thank you for your time. > > Noel W Gray > Neuroscience Graduate Program > SUNY Upstate Medical University > 3219 Weiskotten Hall > 766 Irving Ave > Syracuse, NY 13210-1630 > (315) 464-8144 > [email protected] > _______________________________________________ > Histonet mailing list > [email protected] > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > _______________________________________________ > Histonet mailing list > [email protected] > http://lists.utsouthwestern.edu/mailman/listinfo/histonet >
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