2. Freezing brass planchettes (“hats”), e.g., type B freezing
planchettes.
3. Cryoprotectant: 0.1 M sucrose or 1-hexadecene.
4. 2.0 mL Cryotubes.
5. Tweezers.
6. Liquid nitrogen.
2.3 FreezeSubstitution and Resin
Embedding
1. Cryotubes containing 1.5 mL of 2% OsO4 in anhydrous acetone (prepare in fume hood using gloves and store in liquid
nitrogen) (see Notes 1 and 2).
2. Cryotubes containing 1.5 mL 0.2% glutaraldehyde and 0.2%
uranyl acetate in anhydrous acetone (prepare in fume hood
using gloves and store in liquid nitrogen) (see Note 1).
3. Freeze-substitution/low-temperature resin-embedding system
(e.g., Leica Automated Freeze Substitution device or AFS).
4. Aluminum block with holes for fitting cryotubes.
5. Eponate 12 resin mix without accelerator: 29 g of Eponate
12 resin, 16 g of DDSA, 14.3 g of NMA. It can be kept at
4
C for several days.
6. Eponate 12 resin mix with accelerator: freshly prepared Eponate 12 resin mix with 2.5–3% BMPA (see Note 3). It can be
kept at 4
C for several days.
7. HM20 resin mix: 2.98 g Crosslinker D, 17.02 g Monomer E,
0.1 g Initiator C. Mix the three ingredients in a caramelcolored glass bottle (HM20 is sensitive to light) and keep it at
À20
C.
8. Flat embedding molds.
9. Coverwell imaging chambers (2.8 mm deep; 20 mm diameter).
10. Dry ice and Styrofoam box.
11. Plastic mounting cylinders.
2.4 Preparation of
Sections for Electron
Microscopy
1. Copper/rhodium slot grids coated with 0.25–0.5% (w/v) Formvar in ethylene dichloride.
2. Ultramicrotome.
3. 2% Uranyl acetate in 70% methanol.
4. Reynold’s lead citrate (2.6% lead nitrate and 3.5% sodium
citrate, pH 12).
2.5 Immunolabeling
1. Nickel or gold single slot grids coated with 0.25–0.5% (w/v)
Formvar in ethylene dichloride.
2. Phosphate-buffered saline (PBS): Prepare 1 L of 10Â stock
solution with 1.76 g of NaH 2 PO4; 11.49 g of Na 2 HPO4,
85 g sodium chloride, pH 6.8 (store at room temperature).
High-Pressure Freezing and Freeze Substitution for Transmission Electron. . .
339
planchettes.
3. Cryoprotectant: 0.1 M sucrose or 1-hexadecene.
4. 2.0 mL Cryotubes.
5. Tweezers.
6. Liquid nitrogen.
2.3 FreezeSubstitution and Resin
Embedding
1. Cryotubes containing 1.5 mL of 2% OsO4 in anhydrous acetone (prepare in fume hood using gloves and store in liquid
nitrogen) (see Notes 1 and 2).
2. Cryotubes containing 1.5 mL 0.2% glutaraldehyde and 0.2%
uranyl acetate in anhydrous acetone (prepare in fume hood
using gloves and store in liquid nitrogen) (see Note 1).
3. Freeze-substitution/low-temperature resin-embedding system
(e.g., Leica Automated Freeze Substitution device or AFS).
4. Aluminum block with holes for fitting cryotubes.
5. Eponate 12 resin mix without accelerator: 29 g of Eponate
12 resin, 16 g of DDSA, 14.3 g of NMA. It can be kept at
4
C for several days.
6. Eponate 12 resin mix with accelerator: freshly prepared Eponate 12 resin mix with 2.5–3% BMPA (see Note 3). It can be
kept at 4
C for several days.
7. HM20 resin mix: 2.98 g Crosslinker D, 17.02 g Monomer E,
0.1 g Initiator C. Mix the three ingredients in a caramelcolored glass bottle (HM20 is sensitive to light) and keep it at
À20
C.
8. Flat embedding molds.
9. Coverwell imaging chambers (2.8 mm deep; 20 mm diameter).
10. Dry ice and Styrofoam box.
11. Plastic mounting cylinders.
2.4 Preparation of
Sections for Electron
Microscopy
1. Copper/rhodium slot grids coated with 0.25–0.5% (w/v) Formvar in ethylene dichloride.
2. Ultramicrotome.
3. 2% Uranyl acetate in 70% methanol.
4. Reynold’s lead citrate (2.6% lead nitrate and 3.5% sodium
citrate, pH 12).
2.5 Immunolabeling
1. Nickel or gold single slot grids coated with 0.25–0.5% (w/v)
Formvar in ethylene dichloride.
2. Phosphate-buffered saline (PBS): Prepare 1 L of 10Â stock
solution with 1.76 g of NaH 2 PO4; 11.49 g of Na 2 HPO4,
85 g sodium chloride, pH 6.8 (store at room temperature).
High-Pressure Freezing and Freeze Substitution for Transmission Electron. . .
339