Step | Standard fixation and resin embedding | Hybrid freezing method |
---|---|---|
1 | Prefixed with 2% paraformaldehyde + 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 24 h at 4 °C | |
2 | Vibratome sections at 150 μm, ROI punched out | |
3 | Post-fixation with OsO4 0.1 M cacodylate buffer for 15 min at RT | |
4 | Washing in 0.1 M cacodylate buffer | High pressure freezing with hexadecane as cryoprotectant |
5 | Dehydration in ascending ethanol series | Freeze substitution over 27 h in different substitution cocktails |
6 | Intermedium propylene oxide | Washing in acetone |
7 | Resin infiltration with ascending resin series in propylene oxide | Resin infiltration with ascending resin series in acetone |
8 | Resin polymerization at 60 °C for 72 h in oven | |
9 | Ultra-thin sectioning at 70 nm and stain grids with lead citrate and platinum blue | |
10 | Electron microscopic examination of ultra-thin sections |