[Histonet] How to eliminate holes in mouse brain tissue?

[Geoff McAuliffe]

Hi Amy:

    Your problem is relatively common, you are freezing the tissue too 
slowly. Ice crystals form and when the ice melts, it leaves holes 
behind. You will never get good results by putting the tissue in a 
cryostat at -20C or even -50C , air does not conduct heat  fast enough.
    Freeze the tissue on a rapid freezing stage and/or surround it with 
dry ice 'snow' or partially submerge a metal chuck (with the tissue on 
it) in 2-methylbutane previously cooled with liquid nitrogen or dry ice. 
Then put the tissue in the cryostat and have a cup of coffee while it 
equilibrates to cryostat temp.

Geoff

Amy Janes wrote:

>Hi,
>I am writing to see if I can get some advice on how to eliminate holes I
>am seeing in my tissue.
>I am using tissue from the mouse olfactory bulb and brain for fos ICC.
>Unfortunately I have been unable to see any fos because my tissue is full
>of little holes and therefore I have no actual cells. I have determined
>that it is not the ICC that is causing the holes because they are present
>before staining. So I am assuming I am doing something wrong during
>fixation and cryoprotection. I perfuse the animal with 4% para at a rate
>of 3.5ml/min and I give the animal around 50mls. I then post fix with 4%
>para overnight and then cryoprotect with 30% sucrose until the brain drops
>(usually overnight). The brain is then imbedded in OCT and put in the
>cryostat at -20C to freeze and cut at 20microns. I typically do not
>perfuse with PBS before para but others in my lab have tried perfusing
>with and without PBS and have found no difference in tissue quality. Any
>suggestions as to how I can fix this?
>
>Thanks,
>	Amy
>
>
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-- 
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
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