Fluorescent Larval Fish Feeding Assay

Our procedure was adapted from that described previously [35 (link)]. The larval fish food was fluorescently labeled paramecia prepared using the lipophilic tracer 4-(4-(Didecylamino)styryl)-N-Dethylpyridinium iodide (4-Di-10-ASP; Invitrogen, Carlsbad, CA, USA). We conducted feeding of 7 dpf zebrafish in 6-well plates to allow for free swimming. At 1.5 h after feeding, the larvae were anesthetized. After two washes to remove residual paramecia, groups of five larvae were transferred into a 96-well round-bottom black plate in an anesthetic solution. The intra-abdominal fluorescent signal was measured using the Synergy™ HT Multi-Detection Reader (BioTek Instruments, Winooski, VT) in fluorescence area scan mode 11 × 11 multipoint/well, 0.1 s/point using a fluorescein filter set (excitation wavelength, 485 nm; emission wavelength, 590 nm).