[Histonet] can you defrost and fix muscle tissue?

[Elizabeth Chlipala]

Tyrone

As long as the tissue has been frozen properly you should not have an issue with taking a frozen block to paraffin.  If the tissue has been frozen improperly (slow freezing) then you may encounter some freeze artifact.  We have done it on several occasions without any adverse effects.  We place the frozen sample directly into the fixative rather than thawing and then fixing, replace the fixative and then process as usual.  Sounds more like a processing problem to me, possibly over processed?  Once the blocks were trimmed did you place on wet ice prior to sectioning?  For fixation I would use 4% PFA or 10% NBF.  For staining I would use a modified trichrome one that combines elastic and trichrome staining since you may get disruption of the elastic fibers in atherosclerosis.  We did not work in rat models but we looked at rabbit and mouse models for atherosclerosis, we stained IHC for macrophages, smooth muscle actin and CD31.  Good Luck.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz <@t> premierlab.com
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Friday, October 10, 2014 1:44 PM
To: histonet
Subject: [Histonet] can you defrost and fix muscle tissue?

Hello,

A fellow faculty member had some rat muscle tissue that was set in tissue freezing medium for frozen sectioning. Some sections were cut (just as well as you may realize as you read on) and then the decisions was made to PFA fix and embed in wax. I didn't like the idea as it meant thawing the tissue and possibly introducing freeze-thaw artifacts and compromise the tissue...
But the decision was decided to proceed. The samples were incubated in 4% PFA for 24 hours. The pieces of tissue were about 1.5 to 2 cm (cubic). I took issue with the small volumes of 4% PFA were used (a student was set to do the task) and the volume was greatly increased and incubated for another
24 hours...

We then set about dehydrating, clearing and embedding in wax.

Today, with the student, I tried cutting a few ribbons but it was utterly hopeless. The sections kept crumbling as I cut (thin or thick sections).

Is the issue:
a) fixing frozen tissue
b) improper fixation
c) something else?

To exclude a problem with the wax, some sections of the wax (no tissue) were cut and they were perfect. Regretfully, no fresh muscle was fixed for comparison but that is going to happen if I am asked to repeat this.

For muscle tissue, would you in future suggest PFA or Bouin's? The person is interested in analyzing blood vessels. The rats are hypertensive so I'm thinking Bouin's in case a follow-up study, staining for collagen (scare tissue, artheroclerosis) is desired.

I would like to provide a good explanation for why the sections are crumbling...

Thanks
--
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org
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