Zebrafish Larval SEM Imaging

Scanning electronic microscopy (SEM) was performed as previously described with modifications (Du et al., 2020 (link)). Briefly, embryos after 100% epiboly were treated with 0.06–0.08 g/L PTU (P110661, Aladdin Industrial Corporation, China) to block pigment synthesis. Then larvae at 72 hpf were anesthetized with 0.17 mg/mL^–1 Tricaine (MS-222, Sigma) for 30 s and fixed with 2.5% glutaraldehyde at 4°C over night, followed by post-fixation with 1% osmium tetroxide at 4°C for 2 h. After dehydration in ethanol, samples were critically point dried using a Leica EM CPD300 (Leica, Germany), then mounted and sputter coated with 10-nm platinum using a Cressington 108 sputter coater (Cressington, United Kingdom). Images were taken using a Quanta250 field-emission scanning electron microscope (FEI, Netherlands) with a beam strength of 3 kV.