High-pressure freezing and freeze-substitution for whole cell cultures or thick tissue slices.
Specimens:
- Culture tissue cells on the carbon-coated sapphire disc
- Cell pellets prepared by table-top centrifuge
- Tissue slices by Leica VT1200S automated vibrating microtome and Biopsy punches
Resin options:
- Araldite/TAAB 812 Resin Kit
- Lowicryl HM20
Workflow:
- Place tissue cells in the specimen carrier type-A (Ø3 or 6 mm, 200 µm in depth) filled with 20% BSA in buffer or Matrigel
- Sandwich with the specimen carrier type-B (Ø3 or 6 mm) with preventing tiny air bubbles
- Assemble with the sample holder half cylinders
- Remove an excessive specimen with paper filter
- Vitrification by Leica EM HPM100
- Storage in Cryo-storage Canes or Cryo-EM Pucks in liquid nitrogen dewar (as required)
- Remove the specimen carriers from the sample-holder middle plate in cold pure acetone
- Substitute the frozen specimens into 0.1% tannic acid in anhydrous acetone at -90°C
- Wash in anhydrous acetone at -90°C
- Exchange into osmium tetroxide/uranyl acetate/glutaraldehyde/water mixtures in anhydrous acetone at -90°C, -60°C, -30°C, (up to 4°C as required) by Leica EM AFS2-FSP system
- Wash the specimen with anhydrous acetone
- Infiltrate with a range of Epon/Araldite mixture or Lowicryl HM20 in acetone
- Polymerize at 60°C in TAAB Embedding Oven with Vacuum Lid or Oven Incubators or low-temperature with UV irradiation in Leica EM AFS2