Flash Freezing Protocol for non-perfused rat brains

Posted by Guillermo Palchik on Dec 01, 2008; 3:39pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Acceptor-photobleaching-tp1595536p1599166.html

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

--
There are people who fight one day and are good...
There are those who fight one year and are better...
There are people who fight many years and are very good...
But there are those who fight their entire lives: they are  
indispensable...

Bertolt Brecht (1898-1956)


How can it be that mathematics, being after all a product
of human thought which is independent of experience, is so
admirably appropriate to the objects of reality?
Is human reason, then, without experience, merely by taking thought,
able to fathom the properties of real things?

Albert Einstein (1879-1955)