TEM Grid Staining Protocol

  • Book – Principles in Electron Microscopy – Hyatt

Reagent prep:

Urynl acetate prep– Stored in fridge @ back of EM room (wear gloves)

  • 5ml syringe
  • draw ~ 1.5ml and put on 2um filter
    • expel through filter
    • redraw into syringe without filter
    • replace filter
    • note: Spills can be cleaned with Acetic acid

Lead citrate prep

  • 5mL syringe
  • unclamp lead citrate and cut the tip (to clear a little)
  • pour 1-2mL into tray
  • draw into syringe then add 2um filter
    • expel through filter
    • redraw into syringe without filter
    • replace filter
  • label with white tape

Protocol

  • If staining mouse tissue (this step is not necessary for rabbit)
  • Pre incubate 15 minutes cacodylate buffer
  • 5 minutes (3x) ddH2O (ask Kevin)
  • Be certain to remove all buffers prior to urynal acetate

For all TEM staining:

  1. uranyl acetate (17 mins [not necessary to be exact]) -> cover during this stage
  2. Rinse (3min)
  3. Lead citrate (7min) (make fresh 2 weeks before)
  4. Wash 2x (2min)

(See diagram below for all times)

 

Staining setup diagram

Notes:

  • Cover with plastic box taped @ edge
  • Use another piece of Parafilm to avoid touching base
  • *Do NOT touch Parafilm with finger
  • Let rest at least 1 hour after staining before imaging