2.3. Scanning electron microscopy
For scanning electron microscopy studies, tissue samples cultured on TDZ media were collected at weekly intervals. The collected specimens were prefixed in 2.5% (v/v) glutaraldehyde in 0.1 M Sorensen phosphate buffer overnight at room temperature followed by rinsing with Sorensen phosphate buffer (0.1 M, pH 7.0). The samples were then fixed in 1% (w/v) osmium tetroxide solution overnight at room temperature. This was followed by washing 3–4 times with phosphate buffer and dehydration in ethanol series (50, 70, 80, 90% (v/v) and in absolute ethanol, 10 min per dehydration). Specimens were then critical point dried, mounted on aluminum stubs, sputter coated with gold–palladium and viewed under the scanning electron microscope (Hitachi S-570, Tokyo, Japan) and photographed.