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

[Barbara Schormair]

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
Tel.: 	0049-89-3187-4097
Fax:	0049-89-3187-3474
e-Mail: barbara.schormair <@t> helmholtz-muenchen.de

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