Re: frozen mouse brain

From:Geoff McAuliffe

My experience with frozen storage of mouse brains for immuno is somewhat
similar to yours. We found that the fixed/cryoprotected/OCT brain must be
stored in 2 separate wrapings of heavy duty aluminum foil to prevent
dessication in the -80 freezer. We stored material for only a few months, not 2
years and our freezer did not suffer from the problems yours seems to have.
Still, some loss of immunoreactivity was evident in our material. I suspect
that loss depends on what you are looking for, we were doing tyrosine
hydroxylase in cells which held up well. Fine nerve fibers suffered
significantly, though.
Fresh frozen material, never tried to store it. I would not expect it to look
good even stored at -80.
Sorry I can't be of more help!

Cynthia Favara wrote:

> All,
> I am requesting thought on long term storage of mouse brain. My current
> protocol is to take  one half fresh or NBF fixed,  sucrose protected mouse
> brain usually between 10 and 14 days. Surround with OCT freeze over liquid
> nitrogen and store in -80C.
> Problem is when I go back to these specimens after about 2 years the immuno
> staining seems to be inconsistent. The freezer I use in shared by  a number
> of people and there have been times that the temperature has risen but of
> course there is no documentation or notification [ please don't go there].
> When I stain the sucrose protected specimens I can easily see dried edges
> and the center of the specimen is okay. The fresh frozen sections have
> nonspecific staining throughout the section, and look terrible. Antibody
> being used is a poly clonal in-house anti-gp70. Staining is definitely due
> to primary. Get good staining on specimens less that 1 year in storage.
> I have not done a tremendous amount of work on frozen tissue and just want
> to pick your brains.
> Questions:
> Does anyone have similar experience?
> Do you think the tissue is drying due to freeze/thaw albeit inadvertent?
> Does the high lipid/water content in brain effect storage time?
> Can IHC protocol be identical for frozen vs. paraffin? Of course I do not
> deparaffinize etc.
> Long missive for a curious lady.
> Thanks for your time.
> Cynthia Favara

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

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