[Histonet] Rodent processing and artifactual spaces

[Katherine Murphy]

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 <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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