[Histonet] can you defrost and fix muscle tissue?

[Tyrone Genade]

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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