Imaging and Quantifying Neuromasts and PLLP

Embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄, pH 7.4) overnight at 4 °C. The numbers of cells in the neuromasts and PLLP were counted by DAPI staining as described previously^{18 (link),48 (link),49 (link)}. EdU incorporation was performed as described previously^{47 (link)}. Embryos were dechorionated and 30.5-hpf embryos were immersed in 500 μM EdU solution on ice for 30 min. After washing, embryos were incubated at 28.5 °C for 1 h and fixed at 32 hpf in 4% PFA overnight at 4 °C. The EdU signals were detected using a Click-iT EdU Alexa Fluor488 Imaging Kit (Invitrogen), according to the manufacturer’s instructions. Embryos were embedded in 0.5–1% low melting point agarose (Invitrogen). Confocal images were captured with a Zeiss LSM700 or Zeiss LSM710 microscope and processed using Adobe Photoshop Elements 12 and Fiji^{57 (link)}.