Re: Processing Mouse Tissue

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From:Joyce Kotzuk <>,
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I process mouse brains by hand, and use cedarwood oil and oil of wintergreen (methyl salicylate) instead of xylene for clearing and prior to infiltration with paraffin. The person I learned from said that you must shorten the length of time the tissues are in 100% EtOH, because this is mostly what dries the tissue out. She recommended the following hand processing schedule, which you may adapt to a processor. You could probably still use xylene, because cedarwood oil and methyl salicylate may be cost prohibitive if you're doing large amounts of blocks:
Rinse blocks for 30 mins. to wash out fixative.
3 x 15 mins. 80% ethanol
3 x 15 minutes 95% ethanol
four quick rinses with 100% ethanol
3 x 20 mins. in a 50/50 mix of 100% EtOH/cedarwood oil
1 hour pure cedarwood oil (in a 40 degree oven)
{Could break and leave blocks in cedarwood oil overnight here}
2 x 40 mins. in a 50/50 mix of cedarwood oil/methyl salicylate
3 x 30 mins. pure methyl salicylate
3 x 5 mins. paraffin changes (I use paraplast X-tra)
3 x 15 mins. paraffin changes
several longer changes of paraffin until no wintergreen smell remains
Hope this helps,
Joyce Kotzuk, UNM pathology dept.

>>> Daniel Martinez <> 03/31/00 06:45AM >>>
I am looking for any recommendations with regards to
processing mouse brain tissue.  Our lab currently uses
the same program for processing mouse tissue that is
used for processing tissue from human and other
mammals.  The problem that I have is that when cutting
the tissue it is often to dry to get good sections
without resorting using ammonia water to rehydrate the
tissue every couple of sections.  This can be time
consuming and I would like to add a second program to
our tissue processor that would not dry the tissue
out.  If anyone has done this in their lab and can
recommend a protocol it would greatly be appreciated. 
Thanks for your help.

Daniel Martinez
Center for Neurodegenerative Disease Research
University of Pennsylvania 

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