Suzanne,
	I do sometimes get holes and have not been able to run down the
cause but suspect I am not getting good sucrose penetrationCynthia Favara
Rocky Mountain Laboratories
903 S. 4th Street
Hamilton, MT 59840
406-363-9317
FAX 406-363-9286
. Thanks for the tip I wonder what the comparison between 50% OCT and 30%
sucrose is??? 


-----Original Message-----
From: Suzanne Pham [mailto:Phams@omrf.ouhsc.edu]
Sent: Tuesday, January 30, 2001 12:33 PM
To: histonet@Pathology.swmed.edu
Subject: Re: Cutting fixed brains at 20 microns


Hi,

I have been perfusing mouse brains with 4% paraformaldehyde, soaking
overnight in a 30% sucrose/PBS solution, and then cutting frozen, 20-25
micron sections on a sliding microtome for some time now. It's a fairly
simple method, but there has been the problem of freezing artifact
developing in the tissue, causing holes to form.  I've been experimenting
with a few different conditions to try to get tissue with better morphology
(i.e. no holes at all) and to date, have found that after perfusion,
soaking in a 50% OCT/PBS solution overnight has given me the best results.
The morphology looks better than anything I've gotten with the sucrose
immersion.  I know a lot of people use the sucrose method and I'd like to
know if they get the same holes as I do in their tissues.

Suzanne Pham
Oklahoma Medical Research Foundation



We are asked to cut 20 micron sections of rat brains perfused in a
>paraformaldehyde fixative.  Can you experts out there suggest ways of doing
>this.  I know of adding sucrose to tissue and doing frozen sections. Which
>may be very difficult.  The equipment we have is a tissue tech cryostat and
>rotary microtomes.  Thanks in advance for your help.
>
>Nancy Maronto
>MPI Research
>
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