isopentane freezing of rat brain
m.kirkcaldie at removethis.unsw.edu.au
Thu Mar 3 20:57:32 EST 2005
In article <218d2677.0503030805.2c8d343a at posting.google.com>,
hgpardo at uniovi.es (Hector Gonzalez) wrote:
> I am freezing rat brains using isopentane cooled at - 40 ºC (the
> isopentane bottle is chilled in an ultrafreezer). Usually, I lower the
> brain slowly into the chilled isopentane during aprox. 30 secs in a
> Pyrex glass vase placed in a freezer at -20 ºC. The brain remains
> there for about another 30 secs and then I store it in a freezer at
> -40ºC. Using a cryostate, I obtain 40 micrometer-thick sections at
> -20ºC. However, sections look good, but under the microscope seem to
> be a bit micro-cracked or granulated. I would greatly appreciate any
> tips or suggestions to improve the quality of the brain tissue.
A common method for reducing tissue degradation is to reduce the water
content osmotically - make a 30% sucrose solution in buffered saline,
drop the brain in it, put it in the fridge and wait for it to sink, at
which point the water content has been reduced enough not to rupture
cells when freezing it. If you are using unfixed tissue a tiny amount
of sodium azide (0.1%) can be useful to prevent bacterial growth;
however if you are doing any enzyme-related assays on the sections (e.g.
cytochrome oxidase, succinyl dehrdrogenase, NADPH diaphorase) the azide
will ruin it. You need to wash the sections before any
immunohistochemistry as well, so you would need to use a well subbed
slide like a Superfrost to hold the tissue during that process.
These processes are suitable for fresh or fixed tissue.
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