Thanks for the suggestions. Just a note about the processing protocol...I just recently switched to the lower percentages of alcohol recently to try to remedy the horrible dryness and microchatter I was getting using a 70%, 95% X 2, 100% x 3, xylene x 3 set up where shortening processing times was not solving the problem! Changing the reagent set up solved the microchatter problem but now this 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