frozen mouse brain

From:Cynthia Favara


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. 


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

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