[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.
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
University of Cincinnati College of Medicine
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