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Author Topic: freezing brains for ICC at a later time  (Read 7167 times)

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Offline Maytep

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freezing brains for ICC at a later time
« on: February 03, 2008, 10:09:47 PM »
Some members in the lab have been freezing brains after they sink in 30% sucrose by simply wrapping them in foil and placing them in -80 degrees. They have been seeing small holes in the core of the brains once they thaw out and are sliced. Is there a better way to store them so they don't tear?
One suggestion was to store them in a thick cryoprotectant and keep in -20 and i've also been told to flash freeze using isopentane and keep in -80. Which is better or more common practice?

freezing brains for ICC at a later time
« on: February 03, 2008, 10:09:47 PM »

Offline CanuckPhD

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Re: freezing brains for ICC at a later time
« Reply #1 on: February 04, 2008, 01:35:39 AM »
Placing tissue to freeze in the -80 is the worst way to freeze tissue. I am surprised they are only getting small holes in the middle of the tissue. This freezing artefact is due to the slow and uneven freezing in the -80.

The way to freeze brain is as follows.

1. Sink in 30% sucrose (I prefer 15% but that is up to you)
2. Make a bath of isopentane by placing a beaker of it in a bath of dry ice and abs EtOH. (Do not get any EtOh in the isopentane).
3. Let the isopentane reach -60 (no colder or you tissue will crack BUT no warmer or your tissue will have holes in it)
4. Place the brain into the isopentane for 1 minute and remove.
5. Quickly place the brain on a piece of foil that is on a block of dry ice, wrap tightly and let sit for a few minutes.
6. Then place in the -80

Offline richard03

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Re: freezing brains for ICC at a later time
« Reply #2 on: February 04, 2008, 10:08:21 PM »
I agree with CanuckPhD.

You can NOT simply wrapping them in foil and placing them in -80 degrees.

Re: freezing brains for ICC at a later time
« Reply #2 on: February 04, 2008, 10:08:21 PM »