[Histonet] Frozen mouse embryo
turkekul <@t> gmail.com
Wed Aug 31 10:27:06 CDT 2011
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.
More information about the Histonet