[Histonet] long term storage for IHC

[Pixley, Sarah (pixleysk)]

Here is our method for long term storage for IHC when doing cryostat sectioning, a generous gift from a colleague who kept materials for many years, then did freezing and cryostat sectioning and antibody staining with no losses. Unfortunately, it does take refrigerator space.
This was designed for animal tissues (brain) perfused with 4% paraformaldehyde. I might suggest a washing step(s) in phosphate buffer to go from formalin (which has some alcohol) to the cryoprotectant:

1.	After fixing brain in paraformaldehyde, put directly (no washing) in cryoprotectant
2.	Store in refrigerator for years. 
3.	Remove tissue and place in pure sucrose solution until tissue sinks, at least 24 hours. Can keep in sucrose 1 week or so.
4.	Freeze and section on cryostat.

Cryoprotectant: (30% ethylene glycol v/v; 30 % sucrose, 0.02% Na Azide, 0.04 M Phosphate Buffer)
Total volume: 1 liter: Add, in order indicated:
1.	200 ml dd water
2.	200 ml 0.2 M phosphate buffer (PB) (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 (if you use lower sucrose, make this lower also)
6.	Stir until dissolved
7.	Check volume, but should be 1 liter.
8.	Refrigerate

We used to embed our specimens in our cryomaterial (i.e. OCT), freeze them and kept them frozen in film canisters, similar to what another person suggested. However we kept them at -80 degrees C and we would put a few pieces of regular ice in to make sure they stayed hydrated.  However, sometimes there were air leaks or cracks and the specimens would lose water ("freeze-dry"). As per another suggestion here, I would be wary of using parafilm because it cracks when frozen.  This cryoprotectant method (above) is therefore better than these because of these problems, although the antigenicity is retained when you keep the blocks frozen. 

We do know that you CANNOT keep the frozen sections for very long, because they definitely lose antigenicity. 

Best,
Sarah