Neomycin sulfate hydrate

The repeated neomycin treatments were performed essentially as previously described (Venuto and Erickson, 2021 (link)). At 24 hpf, WT embryos were divided into groups of 30 and grown continuously in the dark at 28.5°C in media supplemented with 200 µM PTU to prevent pigment formation. Larvae were manually dechorionated at 2 dpf. Starting at 9:00 AM, 3 dpf larvae were exposed to either 0 or 50 µM neomycin sulfate hydrate (Alfa Aesar) for 30 min at 28.5°C every 12 h for a total of three treatments. At noon on 4 dpf, treated and untreated groups of larvae were anesthetized with 0.016% MS-222 and fixed in 4% PFA for mRNA ISH. The experiments were repeated three times, each with two technical replicates for the control and treatment groups. Three hours after treatment 1, ablation of lateral line hair cells was assessed by incubating 3 dpf larvae in 130 µM DASPEI (2-[4-(dimethylamino)styryl]-1-ethylpyridinium iodide) for 10 min. Following three washes with E3, larvae were anesthetized with 0.016% MS-222, positioned laterally on a depression slide in 1.2% low melting point agarose, and imaged with a Basler Ace 5.1 MP color camera on a Zeiss SV-11 microscope using an X-Cite 120Q illuminator and wideband GFP filter.