E 1010 ion sputtering device

Sample preparation: The thoracic cavities of the rabbits and the mice were cut open by using scissors and the hearts were removed. After irrigating with phosphate buffered saline (PBS), the hearts were fixed in a solution of 2.5% glutaraldehyde (Res Group Co., Ltd. chemical reagents) for 2 h. Then the atria and ventricles were cut open, and all of the heart valves (including aortic, mitral and tricuspid) were removed. The valves were placed in a freeze-dryer (ES-2030 vacuum freeze-drying device, Hitachi Japan) to be frozen, dehydrated, and dried with tert-butanol for 2–3 h at a temperature of −10 °C.
Characterization of the microstructure on the surface of the heart valve: The heart valves were observed by using the scanning electron microscope (S-3000N scanning electron microscope SEM, Hitachi Japan) at a voltage of 10 kV in order to characterize the microstructures on their surfaces. To improve the conductivity of the sample, before being observed the dried heart valves were treated with spray-gold (E-1010 ion sputtering device, Hitachi Japan) having a coating thickness of approximately 5 nm.

The gross anatomy of the male and female juveniles was observed under an Olympus SZ51 stereomicroscope (Olympus, Shinjuku, Japan). For the scanning electron microscopical observation, the samples were prepared through ethanol gradient dehydration and tert-butyl alcohol replacement. After being frozen for 20 h in -20°C, the samples were dried for 24 h in the Martin Christ Alpha 1-4LD plus freeze-dryer (Martin Christ, Osterode, Germany) before being sprayed with gold by a Hitachi E-1010 ion sputtering device (Hitachi, Tokyo, Japan). The micrographs were taken using a Hitachi S-3400N scanning electron microscope (Hitachi, Tokyo, Japan).