[Histonet] RE: OCT tissuetek

From:"Pixley, Sarah \(pixleysk\)"

Dear Sonny Duong:
It appears that your antigen does not withstand the freezing. That may
be unusual, but I am not surprised - have seen odder things.
I can think of a couple of things to try;
1) Do you save your samples in air-tight containers? i.e. a Zip-loc type
bag? This, plus putting a few pieces of wet ice  into the bag can
prevent freeze-drying from occurring, which could be part of the

2) For long term storage, we now keep our tissues in a cryoprotectant
solution in the refrigerator (then soak them in sucrose and freeze them
one day before cutting). This preserves all of the antigens we have
worked with to date. The person that I got this procedure from has had
preservation of antigenicity for at least a year in some cases.  But
this may not work for you because of the lag between taking out of
cryoprotectant and freezing. See procedure below.

Another hint: After sectioning a frozen block, we thaw the tissue and
reimmerse in sucrose and then if we need the tissue again, we refreeze
and cut. This actually works better than putting the frozen block back
in the freezer and cutting later (it gets too hard and brittle).
Antigenicity of BrdU has not been affected, but we haven't tested
anything else this way.  

3) If you are just waiting for a few days only, then just keep your
tissue in sucrose and freeze just before cutting!

Our general procedure is:
Fix: Perfuse animal (or fix tissues by immersion), post fix for 2-24
hrs, 4% paraformaldehyde.
Rinse with PBS or PB
Immerse in cryoprotectant and store in fridge until needed.
Immerse in sucrose solution (10-30% sucrose in 0.1M phosphate buffer)
for 1-3 days.
Immerse in OCT and freeze on dry ice.
Cut the next day (if we cut the same day, the samples are brittle from
the low temp of the dry ice-but we haven't tried freezing in the
cryostat, which should allow same day cutting).

Cryoprotectant: (30% ethylene glycol v/v; 30 % sucrose, 0.02% Na Azide,
0.04 M PB)
For Total volume of 1 liter, add, in order:
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 until
6.	Stir until dissolved
7.	Check volume, but should be 1 liter.
8.	Refrigerate

Sarah Pixley

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