[Histonet] Thanks for help on "desperately seeking help for cryosectioning" and further question

[Rittman, Barry R]

Barbara
Happy thanksgiving.
1. would recommend that you soak the tissues at step 4 in OCT for some time say half an hour to an hour to allow better penetration of the OCT throughout the tissue. Sounds to me that the OCT has only partly replaced the OCT/sucrose mixture.
 
2. I would recommend that if you are storing at -80C that you includes some ice cubes in the small box or bag that you use to store reduce the risk of dehydrating the block especially on long term storage.
Good luck.
Barry

________________________________

From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Barbara Schormair
Sent: Fri 11/28/2008 8:35 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Thanks for help on "desperately seeking help for cryosectioning" and further question



Dear All,

thank you so much for your suggestions on improving my cryosectioning
results. I tried several of your suggestions, however, the quality of my
sections did'nt improve so much.
I get perfect sections as long as I only cut the OCT embedding media. As
soon as I reach embryonal tissue, only the tissue starts to tear up. In
some of the embryos I loose almost all tissue. In my opinion this can
only be caused by the fixation of the mouse embryos.
Here is our protocol:
1. Fixation in 4% PFA overnight at 4°C
2. 30% sucrose solution at 4°C till embryo sinks to bottom of the tube
3. transfer to 1:1 mix of 30% sucrose solution and OCT for 4h at 4°C
4. transfer to 100% OCT and freeze quickly in isopentane (cooled down
with dry ice for approx. 30min before use).
5. store at -80°C

Is there something wrong? Should I follow a different protocol`?
How long can you store OCT-embedded tissue? Are 6 months too long?

Thanks so much for your help and best regards,

Barbara


--
Dipl. Biol. Barbara Schormair
PhD student
Institute of Human Genetics
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