Does anyone have a TEM protocol for embryos
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Here's what we use for fixing, and processing for TEM, not 100% sure on the protocol after the ultrathins are cut as our EM techs do those bits
Almost all steps are done in glass bijous and on a slow rotating wheel (o/n steps can also be done in the cool room on a rotator).
1. anaesthetise fish and fix immediately in
for about 1.5 h.
2. Depending on the part of the fish you want, try to cut it down after a initial fix of – say – 30min.
3. Wash 3x in Cacodylate buffer.
4. fix in 2% OsO4/Cacodylate for 2 – 3h.
5. Wash in Cacodylate buffer and keep at 4deg in the fridge o/n.
6. dehydrate with 50%, 70%, 80%, 90%, 96% and 100% EtOH over the course of one day. Change 100% EtOH again before keeping the specimen in the fridge o/n. (Can also stop at 90% and continue next day)
7. wash 2x with PPO
8. incubate in 1:1 Epon / PPO o/n.
9. change to 100% EPON
10 change EPON for another o/n incubation
11. embed and harden in 60degrees incubator (1-2 days)
Cut ‘semi-thin’ sections (1-5um) with glass knives on an ultramicrotome
Cut thin sections with a diamond knife