[Histonet] Freezing & storage of rat embryo head/brain

From:"Gale A. Kleven"

I am fairly new to histochemical techniques, and have some tissue storage questions. We are freezing whole rat embryo heads/brains on the fast freeze shelf of a cryostat set to -35 C. Then we place the frozen tissue in vac-u-pac bags, and store at -20 C, with the intent of slicing the specimens within a few months. However, I'm now starting a project where I may not be able to slice and process the tissue (for cytochrome oxidase activity in the brain) for about a year. Concerning long term tissue storage:
1. What are the advantages/disadvantages of long term storage in freezers at -20, versus -40, or -80 C? 
2. Because I'm already storing the specimens in vacuum sealed bags, would freezing & storing in OCT add any benefit? (I have experienced OCT that has become "gummy" after more than a few days when stored in a Ziploc bag at -20 C, but haven't tried it with the vacuum sealed bags.
3. Would storage at colder temperatures necessitate a different freezing protocol (e.g. slower or faster freezing)?
I appreciate any information you can give me on this subject.
Gale A. Kleven
Department of Psychology
University of Iowa                   http://www.psychology.uiowa.edu/
E11 Seashore Hall
Iowa City, IA 52246                 email:  gale-kleven@uiowa.edu

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