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