Eiko ib 5

The samples were cut into 3 × 3 × 2 mm flakes and placed in 4% formaldehyde and 2.5% glutaraldehyde mixed solution (1:1, v/v) for two hours. Then, they were washed with a 0.1 M phosphoric acid salt buffer (pH 7.2) 5–10 times, each before being dehydrated with 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (w/w) ethanol solutions for 10 min. Finally, the samples were dehydrated twice with 100% (w/w) acetone for 10 min each time and then freeze‐dried for 15 hr. The dried samples were mounted on a bronze stub and sputter‐coated with gold (Eiko IB‐5; Hitachi). The microstructures were observed using SEM (Nova Nano SEM 230) at a magnification of 500 times.

For SEM observation, anthers were collected at mature pollen stage and immersed in 3% glutaraldehyde (in 0.1 Mol/L phosphate buffer, pH 7.2) under 4°C for 7 d. The anther samples were then dehydrated with increasing concentration of ethanol series (30%, 50%, 70%, 80%, 90% each for 30 min; 100% twice for a total of 60 min), followed by a final 30-min washing with isoamyl acetate. Before observation in a SEM (S-450, Hitachi, Japan), anther samples were further dried out by critical-point drier (CPD-030, Balzers) and covered with gold using a sputter coater (EikoIB5, Hitachi, Japan) [30 ].

According to Krystyna et al. [24 (link)], the microstructural changes across the muscle fibers were evaluated by a scanning electron microscope (SEM). The 3 × 3 × 5 mm strips, cut along the direction of muscle fibers, were fixed with 3% glutaraldehyde for 48 h. Samples were rinsed with distilled water for 1 h. An ethanol series was used to dehydrate samples. After drying, samples were sputter-coated with gold (Eiko IB-5, Hitachi, Tokyo, Japan). Cross-sections of myofibers were detected with a scanning electron microscope (Quanta 200 FEG, FEI, Eindhoven, Netherlands); the magnification was ×300.