Hi Carmen,
Sometimes people freeze fresh (not fixed) tissues in OCT. When freezing fresh tissues you do not use sucrose. Sucrose is very hyper tonic and will damage live tissue. Alternatively people freeze fixed tissues. Then depending on the stage of the embryo you fix them in neutral buffered formalin or freshly prepared 4% paraformaldehyde for certain time depending on the stage of the embryo. After fixation you wash the fixative with PBS and then place the embryos in 30% sucrose/PBS at 4C. You should keep them in sucrose until they sink to the bottom. If there are air bubbles in the ventricles or other places they will never sink. For embryos bigger than E11.5 days sucrose is overnight at 4C. After sucrose treatment you should incubate the embryos in ice cold OCT for 30 min. Transfer the embryos in fresh ice cold OCT, orient them as you wish and submerge the blocks in iso-pentane (2-methyl butane) pre-chilled in liquid nitrogen to temp of -150C (freezing point of isopentane). Put enough OCT just to cover the embryos and do not keep to long the blocks may crack. Check after 7-10 seconds. It is better to use metal molds lined with aluminium. They conduct heat better. Store the blocks at -80C. Whenever I thaw frozen tissue and freeze it again the morphology is horrible. And with embryos I would not recommend it. Please email me if you need stage specific fixation times. For embryos bigger than E12.5 fix them overnight at room temperature. Best, Mesruh Turkekul mskcc.org _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
