RE: [Histonet] Fixation of frozen tissue?
Thanks George. I have fixed frozen sections before as well in another
lab on a Cryostat, but have never done it at ambient room temp on a
freezing microtome. Can the sections be taken off of the knife with a
paintbrush and placed onto slides at room temp without destroying the
tissue? All of my microtome experience is with free floating sections,
i.e. taking the sections off of the knife and into wells of glycol for
storage until staining, so getting the sections to lay out flat on a
slide wasn't an issue.
Joseph Burdo, Ph.D.
Cellular Neurobiology Laboratory
The Salk Institute for Biological Studies
10010 N. Torrey Pines Rd.
La Jolla, CA, 92037
From: George Cole [mailto:firstname.lastname@example.org]
Sent: Wednesday, February 25, 2004 4:29 PM
To: 'Joe Burdo'
Subject: RE: [Histonet] Fixation of frozen tissue?
In the 6,359 muscles and 2000plus nerves, I always froze the tissues
fresh and sectioned them. I did all fixation and processing on the
fresh frozen sections. You can bolster your brain tissues that you want
to cut by surrounding the tissues with OCT a little at a time, freezing
it as you put it on, to build up a neat block with square edges on all 4
sides around the tissue. You can make sections then, putting the
sections on slides as you go. The quality of the freezing job is
unknown, but brain isn't as bad as muscle for suffering ice crystal
artifact from poor freezing technique. But brain can get a little
difficult to section sometimes. If the brain does look like it's full of
holes, you can try thawing it, then freezing it again, with liquid
nitrogen and isopentane---this worked with muscles. Fixation after
freezing was successful in my lab about 120,000 times. The freezing job
on the brain as it comes to you is the unknown here. All you can do is
cross your fingers and try to go ahead. I hope it works all right.
[mailto:email@example.com] On Behalf Of Joe
Sent: Wednesday, February 25, 2004 3:20 PM
Subject: [Histonet] Fixation of frozen tissue?
I have some dry ice frozen human brain samples that I would like to
section for IHC. We do not have a Cryostat in our lab, but we do have a
freezing microtome. Can the frozen brain chunks be fixed in 4% PF,
cryoprotected in 30% sucrose and then sectioned on the microtome for
free floating IHC? I have never cut tissue that has been fixed after
freezing, only tissue that has been fixed immediately after removal.
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