- Fix tissues/cells for
only one hour in cryofix.
- Spin down in small
table top centrifuge at about 850 x g.
- Resuspend cells in 3%
liquid gelatin. Place on ice for 10 min.
- When gelatin is solid,
add more cryofix.
- After 15 minutes,
loosen pellet from the sides of the tube.
- Fix for 15 more
- Place in 2.3 M sucrose
with PVP solution.
- Leave overnight in
- Collect 2 pairs of
forceps, cryo storage tubes, clean screws and liquid N2.
Freezing of Tissue for
- Label the cryotubes
with EM #, date, PI, and specific sample #.
- Bore holes into the top
half of the tube with a needle and place the tube in liquid N2.
- Pour sucrose/tissue
mixture into petri dish.
- Mince pellet into 0.5
- Freeze the blocks one
at a time, one block per screw, by immersion in liquid N2.
- Place the screw in the cryo tube
immersed in liquid N2.
- It is now very important that the
tissue remain frozen.
- Store in R2D2.
- 1 x PBS [Phosphate
Buffered Saline, 10X Solution, Fisher Scientific]
- 2.5% Glutaraldehyde in
PBS [25% Taab Glutaraldehyde, EM Grade, Energy Beam Sciences]
- Cryofix Fixative(2%
Paraformaldehyde; 0.01% glutaraldehyde in PBS)[ Formaldehyde, Para,
Certified, Fisher Scientific]
- 2.3 M Sucrose in PBS
[Sucrose, Fisher Scientific]
- 20% PVP 1.6 M Sucrose
buffered with 0.055 M Sodium Carbonate [polyvinylpyrrolidone and Sodium
Carbonate, Fisher Scientific]
- 1% Osmium Tetroxide
(OsO4) in dH2O [Osmium Tetroxide, Electron Microscopy Sciences]
- 1% Osmium Tetroxide
(OsO4) + 1 % Potassium Ferricyanide [Potassium Ferricyanide, Fisher
- Ethanol (30% in PBS,
50% in PBS, 70% in dH2O, 95% and 100%) [(30- 95%) Reagent Alcohol,
Fisher Scientific, and 100% requisitioned in pints]
- Propylene Oxide
[Propylene Oxide, Electron Microscopy Sciences]
- Epon Resin [Scipoxy
812, DDSA, NMA, and DMP-30, Energy Beam Sciences]
- LR White Resin [LR
White Resin, Electron Microscopy Sciences]
- 0.5% Toluidine Blue in
1% Sodium Borate [Toluidine Blue O Certified Biological Stain and
Sodium Borate, Fisher Scientific]
- Sodium Hydroxide
[Sodium hydroxide pellets, Fisher Scientific]
- Reynold’s Lead
Citrate [Lead Nitrate and Sodium Citrate Dihydrate, Fisher Scientific]
Uranyl Acetate [Uranyl Acetate, Electron Microscopy Sciences]