TABLE 1.
IRF protocol with freeze-substitution steps after transfer of samples from QFS-box to Leica AFS2 freeze-substitution apparatus. QFS,quick-freeze substitution.
| Reagent | Temp | Time | Repeats |
|---|---|---|---|
| Acetone | −50°C | 20 min | 2x |
| Ethanol | −50°C | 20 min | 1x |
| 20, 40, 60, 80% HM20/ethanol | −50°C | 45 min | each step 1x |
| 100% HM20 | −50°C | 8 h | 1x |
| 100% HM20 | −50°C | 2 h | 3x |
| 100% HM20 - UV polymerization | −50°C | 48 h | 1x |
| 100% HM20 - temperature rise with UV polymerization | −50 - 20 °C | 16 h | 1x |
| 100% HM20 - UV polymerization | 20°C | 48 h | 1x |