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,
Dipl. Biol. Barbara Schormair
Institute of Human Genetics
Helmholtz Zentrum München
German Research Center for Environmental Health (GmbH)
Ingolstaedter Landstraße 1
Chairman of Supervisory Board: MinDir Dr. Peter Lange
Board of Directors: Prof. Dr. Günther Wess and Dr. Nikolaus Blum
Register of Societies: Amtsgericht München HRB 6466
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