Figure 1 shows the flow of the sample preparation procedure for TEM and FE-SEM observations.
- Fixation and embedding
1. Pre-fixation: 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 3 h at 4 °C.
2. Rinse: 0.1 M phosphate buffer (pH 7.2) for 10 min three times at 4 °C.
3. Post-fixation: 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4 °C.
4. Dehydration: ascending ethanol series, 60%, 70%, and 80% at 4 °C, followed by 90% and 95% at room temperature (RT) for 10 min each, and 100% at RT 2 times for 10 min each.
5. Substitution: propylene oxide for 10 min, two times at RT.
6. Substitution: 1:1 Epon812 epoxy resin and propylene oxide overnight at RT
7. Embedding: Epon812 for 3 h at RT, and then Epon812 for 2 days at 60 °C.
8. Thin sections: 80-nm thin sections on the grids for TEM and 200- to 500-nm semi-thin sections on the slide glasses or silicon substrate for FE-SEM, prepared with an ultramicrotome.
- EM staining
1. Pretreatment: MH was filtered through a 0.45-μm Millipore filter before use.
2. Pre-staining: Samples were stained with MH for 10 min for TEM and 20 min for FE-SEM at RT3).
3. Wash: Double-distilled water for 3–10 min.
4. Post-staining: RPb solution4) for 5 min at RT.
5. Wash: Double-distilled water for 1–3 min.
6. Osmium conductivity coating for FE-SEM
The samples were observed and recorded using a TEM or an FE-SEM attached to a backscatter electron detector.