[Histonet] Rodent processing and artifactual spaces

[Jackie O'Connor]

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.murphy <@t> gmail.com>
To: Elizabeth Chlipala <liz <@t> premierlab.com>
Cc: Jackie O'Connor <b427297 <@t> aol.com>; rjbuesa <rjbuesa <@t> yahoo.com>; histonet <histonet <@t> 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 <liz <@t> 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-bounces <@t> lists.utsouthwestern.edu
 [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jackie
 O'Connor
 Sent: Thursday, August 02, 2012 11:36 AM
 To: rjbuesa <@t> yahoo.com; katherine.o.murphy <@t> gmail.com;
 histonet <@t> 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 <rjbuesa <@t> yahoo.com>
 To: Katherine Murphy <katherine.o.murphy <@t> gmail.com>; histonet
 <histonet <@t> 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.murphy <@t> gmail.com>
 o: histonet <@t> 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?
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