Re: cracked brain blues

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From:"Harclerode, Donna" <>
To:"''" <>, "''" <>
Date:Wed, 21 Jul 1999 08:25:02 -0700

Hi Bruce,
You did not say how you are sectioning the rat brain, but isopentane is not
necessary for cryoprotected brains.  Freezing more slowly using only dry ice
works very well for sliding microtome and cryostat work. I used fresh 30%
sucrose to attach the brain to the stage for the sliding microtome or
cryostat chuck.
When I worked in Neuroscience my standard protocol was to perfuse with 4%
PFA, post fix overnight then transfer to 30% sucrose for at least 24 hours.
After sectioning with sliding microtome the sections (20-50u, usually) were
processed free-floating for IHC.  Cryostat sections (4-10u) were picked up
and processed on slides for IHC.
I have had fresh frozen blocks crack using OCT (frozen embedding media)
sometimes when I left them too long is isopentane. (I am not sure what M-1

Hope this helps

Donna Harclerode HT (ASCP) HTL, QIHC
Ligand Pharmaceuticals
San Diego, CA

>Hi histoneters,

>I have been getting major cracking when freezing rat brains lately.
>History: perfusion with 4% parafomaldahyde, post-fixation 4%
>parformaldahyde one hour, cryoprotection 20% sucrose in PB 18hrs. Brains
>were coated lightly with Lipshaws M-1 then dropped into isopentane which
>was cooled with dry ice.  Where did I go wrong?
>Thanks in advance
> --------------------------------------
>Bruce Rasmussen
>Predoc Fellow in Experimental Neuropsychology
>he Krasnow Institute for Advanced Study
>George Mason University
>Mail Stop 2A1
Fairfax, VA 22030-4444
- ---------------------------------------

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