High-pressure freezing and freeze-substitution.

High-pressure freezing and freeze-substitution for whole cell cultures or thick tissue slices.

Specimens:

  1. Culture tissue cells on the carbon-coated sapphire disc
  2. Cell pellets prepared by table-top centrifuge
  3. Tissue slices by Leica VT1200S automated vibrating microtome and Biopsy punches

Resin options:

  1. Araldite/TAAB 812 Resin Kit
  2. Lowicryl HM20

Workflow:

  1. 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
  2. Sandwich with the specimen carrier type-B (Ø3 or 6 mm) with preventing tiny air bubbles
  3. Assemble with the sample holder half cylinders
  4. Remove an excessive specimen with paper filter
  5. Vitrification by Leica EM HPM100
  6. Storage in Cryo-storage Canes or Cryo-EM Pucks in liquid nitrogen dewar (as required)
  7. Remove the specimen carriers from the sample-holder middle plate in cold pure acetone
  8. Substitute the frozen specimens into 0.1% tannic acid in anhydrous acetone at -90°C
  9. Wash in anhydrous acetone at -90°C
  10. 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
  11. Wash the specimen with anhydrous acetone
  12. Infiltrate with a range of Epon/Araldite mixture or Lowicryl HM20 in acetone
  13. Polymerize at 60°C in TAAB Embedding Oven with Vacuum Lid or Oven Incubators or low-temperature with UV irradiation in Leica EM AFS2