Re: Flash Freezing Protocol for non-perfused rat brains

Posted by Stephen Cody-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-photobleaching-tp1595536p1620588.html

I have not worked with brain tissue but....

 

Don't immerse directly into liquid liquid nitrogen (LN2) it will rapidly boil, the LN2 bubbles will very effectively insulate the tissue, and the cooling rate will be very very slow. Gaseous nitrogen is a very effective insulator.

 

Try using a modification of the method described by Pertti - cool the isopentane in a polyethylene plastic beaker immersed in liquid nitrogen, stir with a wooden stick until some of the isopentane starts to freeze on the bottom of the beaker. This will give rapid cooling, the faster the cooling he smaller the ice-crystals. The smaller the ice crystals at the surface of the tissue, the better the tissue will be deeper in the thick slice. Ice crystals will grow bigger and bigger deeper into thick tissue, so the faster the freezing the better.

 

Also try using a cryoprotectant such as OCT, but just a thin layer. Don't allow the tissue to thaw; always pre-cool the forceps before removing the tissue.

 

Cheers

Stephen Cody 

2008/12/2 Pertti Panula <[hidden email]>

Hi,

We have been doing this for many years for in situ hybridization. We use a 50 ml plastic tube filled with isopentane. This is placed in alcohol in a beaker. Alcohol is precooled with pieces of dry ice. The exact amounts needed are difficult to tell, we follow the freezing speed. We keep adding dry ice as it melts. Freezing of whole rat and mouse brains works fine. Do not leave the brains in isopentane too long, remove them from the tube with forceps as soon as they are frozen. Then keep them at -20--80 until cutting. Before cutting allow enough time in the cryostat to get the sample in appropriate cutting temp.

Feel free to write off the list if you need more details.

Best regards Pertti Panula

Guillermo Palchik wrote:

Dear Confocalists,

Does anybody have a good working protocol for flash freezing fresh rat brains (not perfused) ?
I have been doing flash freezing by cooling Isopentane to -50 C and then scooping the fresh brains into it and letting it sit for about 5-10 seconds, then scooping it into dry ice an letting it sit for a few minutes and then into the -80 freezer.
The problem is that I have many cracks (crystals) when I cut the brains in the cryostat.
After doing some research I found out that proper flash freezing is supposed to prevent that...
I have tried leaving it longer than 20-30 seconds in the Isopentane (-50C) but the brains crack open.
I also tried directly placing them into LN2 but it cracks after 5 seconds...
I have heard that cooler Isopentane (-80 C) might work.
Any ideas?
Thanks
Gil Palchik

--

Pertti Panula
Professor, Research Director
Neuroscience Center
Institute of Biomedicine/Anatomy
POB 63
00014 University of Helsinki
Finland
Phone:  [phone] 
Fax: [phone]
Mobile:  [phone] 
[hidden email]
http://www.helsinki.fi/neurosci/panula.htm

--
Stephen Cody