Just got to thinking...I do animal tissue processing. I do use an automated processor, but it is open with no pressure or vacuum. If you think this will help I can give you the times I use. It is an overnight process and takes more than a work day worth of hours. Let me know?
Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Margaret Blount Sent: Tuesday, February 22, 2011 3:02 AM To: Noel Gray; [email protected] Subject: RE: [Histonet] Formalin-fixed paraffin embedded question 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 _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
