AMy, 

Freezing cryoprotected tissue in a cryostat will create freezing artifact
(big holes caused by water ice crystal formation with lower
temperature/slower freezing in a cryostat).  You need to SNAP FREEZE.
Histonet archives is filled with suggestions on various ways to snap
freeze, do a search as this has been discussed at length recently. Sucrose
helps reduce this problem, but it can still rear its ugly head with
cryostat freezing.  

Be sure you cut at a correct temperature also, cryoprotected/prefixed
tissue I find are cryosectioning well at around -26C otherwise the sucrose
oozes out like Karo syrup. 

  At 02:11 PM 1/21/2004 -0500, you 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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>
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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