Hello Caroline,
You did not mention what animal tissue you'll be using. In any case, say for a large
animal brain like primate why not perfuse with saline (no fixation solution), and remove
the brain. Decide how thick (in microns) you want the fresh brain to be blocked e.g. 3mm.
Then just take every other one tissue block and freeze using dry ice in isopentane and corretly
store these blocks in -80C until ready to cut fresh frozen sections on either the sliding
microtome or cryostat. You have to decide on the thickness of these sections e.g. 20ums.
Then the other blocks NOT for fresh frozen sections can be placed in a fixative solution
of you choice. Immersion fixation in a solution for - overnight at 4C with gentle agitation
(next day check tissue to make sure its fixed - no pink color on tissue). After tissue blocks
have been fixed, wash 2X in PBS (agitage) @ RT to remove residual fixative solution - 20 mins
each wash. Then place these tissue blocks in 30% sucrose made in PBS - @ 4C w/agitation
until the tissue sinks to the bottom of container - takes a few days.
After this has been done, pad each block of excess sucrose solution with kimwips and freeze
brain section blocks (see histonet archives for freezing tissue using aluminum block method
& liquid nitrogen or if you are interested in this method I will provide a protocol...later). Then
store each block properly in -80C freeze until ready to section for fixed free-floating sections
@ 40ums.
I hope this helps some.
best regards
Maria Bartola Mejia
Department of Neurosurgery
UCSF
SF CA 94103
-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu on behalf of Caroline Bass
Sent: Tue 8/14/2007 9:46 PM
To: Histonet
Subject: [Histonet] quick and easy sectioning of fresh brain
Hey Folks,
So I want to have my cake and eat it too. I have injected a rat with
a virus that will express EGFP. Ideally I'd like to collect the
fresh brain, cut off thick floating sections, enough to get a feel
for the spread of the virus, and possible punch out the EGFP area to
get mRNA and/or protein for qPCR and western blot. Are there any
suggestions for doing this? I have access to cryostats, sliding
microtomes, and brain molds. Cutting fresh brain is difficult, so
I'd like to freeze it to section on the sliding microtome, collect a
floating section, and visualize under a fluorescent dissecting
microscope. Are there any issues with doing this? How should I
freeze the brain, and how will this affect mRNA and protein levels?
How difficult are unfixed floating sections to deal with? Finally,
can I take some sections and fix by immersion in PFA so that I can
store for longer periods of time.
Any suggestions would be appreciated. I've had very good luck
working with perfused/fixed tissue, and would like to continue with
it, but it doesn't seem possible with the mRNA/protein endpoints.
Thanks,
Caroline
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