Deep-etch EM

Deep-etch, or freeze-fracture EM for plasma membrane (membrane skeletons, clathrin-coats, caveolae), other membrane compartments, and cytoskeletal organizations [under construction].

Workflow for Deep-etch EM:

  1. Culture tissue cells on small glass coverslips
  2. Wash with Mammalian Ringer solution (155 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM NaH2PO4, 5 mM HEPES brought to pH7.4 with NaOH, plus 10 mM glucose) or Hank’s balanced salt solution
  3. Treat with medium molecular weight poly-L-lysine in the Ringer solution
  4. Unroof the plasma membrane by ultra-gentle sonication
  5. Chemical fixation with half-strength Karnovsky solution (2% paraformaldehyde - 2.5% glutaraldehyde) in KHMgE buffer (30 mM HEPES buffer brought to pH7.4 with KOH, 70 mM KCL present to make it roughly isotonic to the intracellular milieu, 5 mM MgCl2, 3 mM EGTA present to eliminate calcium)
  6. Wash with KHMgE buffer and Rinse with pure water
  7. Rapid freeze with pure copper metal cooled in liquid helium
  8. Deep etch at minus 90-80°C
  9. Rotary shadow at 22° with pure platinum and carbon
  10. Wash with hydrofluoric acid and pure water (including Kodak Photo-Flo 200)
  11. Mount on the carbon-coated Formvar grid
  12. TEM imaging

Workflow for Freeze-fracture EM:

  1. Culture tissue cells on small glass coverslips
  2. Rapid freeze with pure copper metal cooled in liquid helium
  3. Freeze fracture at minus 103°C
  4. Rotary shadow at 22 with pure platinum and pure carbon
  5. Wash with hydrofluoric acid and pure water (including Photo-Flo 200)
  6. Mount on the carbon-coated Formvar grid
  7. TEM imaging

*Leica freeze-fracture system (Ø4.6 x 0.6 mm)

Immune-gold labelling options:

  1. SDS-FRIL (SDS-digested freeze fracture replica immunogold labelling)
  2. DRIL (Deep-etch replica immunogold labelling