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Cagan Lab - Protocols

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EM fixation:

  1. 1.dissect in PBS

  2. 2.fix in PLP for 30’

  3. 3.Wash 3X10’ in PBS

  4. 4.fix 30’ in 2% glutataraldehyde (from freezer) in PBS

  5. 5.wash 3X 10’ in PBS
    post-fix in OsO4/K3Fe(CN)6 for 60’
    add equal volumes of OsO4 and K3Fe(CN)6 from tubes in freezer container marked “Microscopy”.

  6. 6.Wash 3X10’ in PBS

  7. 7.Dehydrate in EtOH series: 30%, 50%,70%,80%,95%, and 3X100%
    !!!!!!!!!No cheating!!!!!!!!!!!!!! This is critical for EM!!!!!!!

  8. 8.3X10’ in propylene oxide

  9. 9.overnight in 1:1 propylene oxide:EM resin
    use gloves

  10. 10.5 hrs in 100% resin; imbed