Smz18 stereomicroscope

Stained fish skeletons were imaged using a Nikon SMZ18 stereomicroscope and NIS-Elements software. Adult fish were anesthetized with Tricaine mesylate (MS-222; 0.04%, Western Chemical Inc.) and photographed using a Canon EOS digital camera. Imaging of live and stained larval fish were performed using a Zeiss LSM 800 confocal microscope and ZEN Blue software or a Nikon SMZ18 stereomicroscope and NIS-Elements software for low magnification images of whole larvae. Larvae were anesthetized with MS-222 and embedded in 0.5% low-melting agarose for imaging. Confocal z-stacks were processed using ZEN blue software.

The stem trichome images were acquired using the Nikon SMZ18 stereo microscope equipped with the DS Ri2 colour camera and run by the NiS Elements AR software. The total image magnification was 15x. For scanning electron microscopy (SEM), leaf pieces were fixed for at least 1 h under vacuum while submerged in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate at room temperature, then stored overnight at 4 °C. Samples were rinsed in buffer, postfixed in 1% osmium tetroxide in buffer overnight at 4 °C, rinsed in ultrapure water (NANOpure Infinity®; Barnstead/Thermo Fisher Scientific; Waltham, Maryland), and dehydrated in an ethanol series. Samples were then processed in a critical point dryer (Autosamdri-814; Tousimis; Rockville, Maryland). Material was mounted on aluminum stubs using double-sided adhesive carbon tabs and colloidal graphite, sputter-coated with gold-palladium, and observed in a scanning electron microscope (S3500N; Hitachi High Technologies America, Inc.; Schaumberg, Illinois) at an accelerating voltage of 10 kV.