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  1. Tamara AUTHOR

    Does anyone have a TEM protocol for embryos

    1. Hi Tamara,

      You may also want to send this question to the Zebrafish Newsgroup:

      http://zfin.org/zf_info/news/newsgroup.html

      It's easy to join the Google Group and the list members from the zebrafish research community can be very helpful in answering questions such as this one.

    2. 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

      TEM – starting protocol for zebrafish

      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).

      Day1

      1. anaesthetise fish and fix immediately in

                  4% Glutaraldehyde

                  1% PFA

                  0.05M Cacodylate

                  1mM MgSO4

                  1% Sucrose

      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.

      Day2

      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)

      Day3

      7. wash 2x with PPO

      8. incubate in 1:1 Epon / PPO o/n.

      Day4

      9. change to 100% EPON

      10 change EPON for another o/n incubation

      Day5

      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