[Histonet] RE: Fixative for storage of perfused rat organs

From:"Pixley, Sarah (pixleysk)"

Dear Clover Daley:
Depending on your antigens, I would not store in paraformaldehyde-too harsh.
For storage, you can store short periods of time in phosphate buffer with
sodium azide (i.e. 0.02%). You definitely need to refrigerate and add azide
because otherwise they will get bacteria and/or mold. If you are going to do
frozen sectioning, then I currently use and really like the following
protocol for long-term storage. Supposedly this allows storage for years
without loss of antigenicity. (If you are paraffin embedding, store the
tissues after embedding in paraffin.)

Cryoprotection and long-term storage:
Preparing tissues for long-term storage prior to cryostat sectioning.
*	Perfuse or post-fix tissues in paraformaldehyde.
*	Rinse with buffer or water
*	Decalcify if necessary, rinse with water, then buffer.
*	Put tissues in Cryoprotectant (see below), Store in fridge until
needed: supposedly good for years.
*	Before sectioning, put tissues in 30% sucrose in 0.1M PB, overnight
or until tissue floats. Can keep in sucrose 1 week or so.
*	Remove tissue, embed in M1 embedding matrix (Shandon).
*	Freeze tissue in cryostat, cut cryostat sections, discard uncut

Cryoprotectant: (30% ethylene glycol v/v; 30 % sucrose, 0.02% Na Azide, 0.04
Total volume: 1 liter: Add, in order indicated:
1.	200 ml dd water
2.	200 ml 0.2 M phosphate buffer (final conc. 0.04M PB)
3.	300 ml Ethylene Glycol
4.	10 ml of 2% Na Azide
5.	300 g sucrose: add slowly (~100 g at a time) with stirring
6.	Stir until dissolved
7.	Check volume, but should be 1 liter.
8.	Refrigerate

30% Sucrose in 0.1M Phosphate Buffer: For 500 mls:
1.	250 mls 0.2 M PB
2.	150 g sucrose
3.	100 mls water
4.	5 mls 2% Na Azide (100X)
5.	Stir until dissolved
6.	Add water to make up to 500 mls
7.	Refrigerate.

Sarah Pixley
University of Cincinnati College of Medicine

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