We trim our tisues the day after necropsy, put the cassettes back in formalin for a few hours on the processor before it starts. Our sections are durn perfect, if I do say so myself. One of the things I have found important for rodent tissues is to face the blocks and leave them on wet ice for about an hour before taking sections. No chatter whatsoever. We routinely cut at 5 microns.
Jackie O' -----Original Message----- From: Katherine Murphy <katherine.o.mur...@gmail.com> To: Elizabeth Chlipala <l...@premierlab.com> Cc: Jackie O'Connor <b427...@aol.com>; rjbuesa <rjbu...@yahoo.com>; histonet <histonet@lists.utsouthwestern.edu> Sent: Thu, Aug 2, 2012 1:39 pm Subject: Re: [Histonet] Rodent processing and artifactual spaces Thanks for the suggestions. Just a note about the processing rotocol...I just recently switched to the lower percentages of lcohol recently to try to remedy the horrible dryness and icrochatter I was getting using a 70%, 95% X 2, 100% x 3, xylene x 3 et up where shortening processing times was not solving the problem! hanging the reagent set up solved the microchatter problem but now his other separation artifact has appeared. On 8/2/12, Elizabeth Chlipala <l...@premierlab.com> wrote: I agree with Jackie, I process from 20 to 30 minutes a station for mouse and rat tissue, but I process 45 minutes per station for brain and for skin I process one hour per station. I find that once you gross the tissue samples in they need to be placed back into formalin to fix additionally and I never gross mouse or rat tissue and place in 50 to 70% alcohol that just causes problems, that’s been my experience. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308-1592 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell www.premierlab.com Ship to address: 1567 Skyway Drive, Unit E Longmont, CO 80504 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jackie O'Connor Sent: Thursday, August 02, 2012 11:36 AM To: rjbu...@yahoo.com; katherine.o.mur...@gmail.com; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Rodent processing and artifactual spaces I think the processing times are fine, sorry Rene'. They are pretty similar to my processing schedules for both species. It could be more of a fixation artefact as you suspect. Tissues will begin to degrade waiting for formalin to get to them, and although formalin most tissues at about 3mm per hour - whole organs tend to slow penetration down somewhat. The low volume of fixative to tissue only compounds the problem. Jackie O' -----Original Message----- From: Rene J Buesa <rjbu...@yahoo.com> To: Katherine Murphy <katherine.o.mur...@gmail.com>; histonet <histonet@lists.utsouthwestern.edu> Sent: Thu, Aug 2, 2012 12:03 pm Subject: Re: [Histonet] Rodent processing and artifactual spaces Rat and mice tissues do not have too much fat and are susceptible to dryness fter processing specially when using xylene as "clearing" agent. also think that your times are too long. he best processing protocol for rodent tissues is a sequence of 2-propanol → ropanol+mineral oil → mineral oil → paraffin. t has produced great results in those labs that are using it. f you are interested I can send you under separate cover the procedure. ené J. _______________________________ rom: Katherine Murphy <katherine.o.mur...@gmail.com> o: histonet@lists.utsouthwestern.edu ent: Thursday, August 2, 2012 11:07 AM ubject: [Histonet] Rodent processing and artifactual spaces Hello Histo Gurus, I have a question for you: I process rat and mouse tissues (kidney, liver, lung, brain, pancreas, I, spleen) and have been seeing a separation artifact in the tissues esp. kidney, brain, and heart) on the slides. I believe this is what as described as artifactual spaces between individual cells or cell hrinkage, as described by Carson (1997) under Fixation and rocessing. The processing schedule that has produced this is: 50% eagent Alcohol (RA), 70% RA, 80% RA, 95% RA x 2, 100% RA x 2, xylene 2, and Paraplast Plus @ 60°C with vacuum, at 40 min per station for at and 25 min per station for mouse. Preceding processing is a 20’ ap water rinse. Tissues have been in formalin for weeks in most cases ut not always collected ideally (low formalin to tissue ratio, small ars used for large tissues, tissues submitted whole without slices or cores, etc)--we don't have much control over this part. Does anyone now what causes the separation artifact is and how it can be orrected? _______________________________________________ istonet mailing list isto...@lists.utsouthwestern.edu ttp://lists.utsouthwestern.edu/mailman/listinfo/histonet ______________________________________________ istonet mailing list isto...@lists.utsouthwestern.edu ttp://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet