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Osmium Textroxde (OsO4) Protocol

This is designed to add "mass thickness" to tissue by decorating
membranes and strucures with heavy atoms. OsO4 is highly
toxic. Know what you are doing. Also, you can over-osmicate, making the
tissue brittle, non-immunoreactive, and too dense/contrasty for imaging.
Finally, osmium diffuses slowly and is poorly water soluble, so tissue pieces
should be less than 1 mm in smallest dimension. Retina is fine.

Stocks

  • 2% OsO4 in deionized water protected from light.
    • Rinse a teflon-stoppered 100ml volumetric flask 3x with 0.1 N HNO3 and 3x with dH2O. Add 50 ml dH2O,
    • Wash a 1 gram vial of OsO4 in warm soapy water.
    • Wear goggles, apron, nitrile gloves and lay out paper towels in the hood.
    • Place the vial in a beaker with hot tap water until the OsO4 crystals melt.
    • Score the vial neck with a small trangular file, grasp wrapped in paper towels, break and drop into the volumetric flask.
    • Sonicate until dissolved (several hours)
    • Store wrapped in foil in hood.
  • 0.2 M phosphate buffer PB or cacodylate buffer PB pH 7.4
  • 0.1 M phosphate buffer PB or cacodylate buffer PB pH 7.4

Procedure

  1. In hood, combine 1cc OsO4 stock and 1 cc 0.2 M PB and CB stock in a glass scintillation vial.
  2. Rinse fixed tissue 2 x 10 min in 0.1 M PB or CB.
  3. Add 1% OsO to tissue just to cover4
  4. Osmicate 30-60 min (cold or RT as needed).
  5. Rinse fixed tissue 2 x 10 min in 0.1 M PB or CB.
  6. Proceed to UA or dehydration.

NOTES:

Discard waste in the Osmium Waste bottle in the hood. Call ESH for pickup.

MSDS: JT Baker Osmium MSDS



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