Scanning electron microscopy (SEM)
| Materials |
|---|
| Fixation buffer: 3% Formaldehyde, 2% Glutardialdehyde, 10 mM CaCl2, 10 mM MgCl2, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |
| Washing Buffer: 10 mM CaCl2, 10 mM MgCl2, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |
| HEPES buffer: 100 mM, pH 7.2 |
| Graded ethanol series: 30%, 50%, 70%, 90%, 100% ethanol p.a. |
Procedure:
1A. Organs of infected animals are excised, washed with PBS, and incubated in cold fixation buffer over night at 4°C. (Time depends on size and texture of tissue)
1B. Isolated cells grown on glass coverslips are fixed by gentle pipetting of 1 ml of cold fixation buffer directly onto the side of the coverslip, thereby slowly pushing the cell culture medium up, creating a layer of fixative underneath the cell culture medium. Do not mix at this point ! Incubate for 5 min at RT, then aspirate medium and fixative and add 1 ml of fresh fixation buffer for at least 30 min at RT. Samples can be stored before further processing in fixative at 4°C.
(Note: Used fixation buffer should be collected in a separate waste bottle!)
2. Fixed samples are washed 3 times with washing buffer (3 x 10 min)
Optional: 1% OsO4 in dH2O for 60 min at 4°C
Samples are washed 3 times in 100 mM HEPES pH 7.2 (3 x 10 min).
3. Dehydration in a graded series of ethanol:
30% ethanol 4°C, 7 min
50% ethanol 4°C, 10 min
70% ethanol 4°C, 10 min (storage overnight at 4°C is possible at this step)
80% ethanol RT, 10 min
90% ethanol RT, 10 min 3x100% ethanol RT, 10 min
(For tissues use 60 min incubation times)
4. After critical point drying from liquid CO2, samples are sputter-coated with 5 nm gold-palladium in a BAL-TEC SCD 030 and examined at 15 kV accelerating voltage in a Zeiss Auriga or Zeiss Evo scanning electron microscope using the secondary electron detector.
After critical point drying from liquid CO2, 8 Zyklen a 10 min, Enddruck: xx bar@ 44°C, samples are sputter-coated with 6 nm Pt and examined at 15 kV accelerating voltage in a Zeiss Auriga or Zeiss Evo scanning electron microscope using the secondary electron detector.