Bodipy c 5

Fifteen larvae per treatment at 8 dpf were placed into 12-well plastic dishes (3 larvae per well) and soaked in 100 μM BODIPY C-5 (4,4-Difluoro-1,3,5,7-Tetramethyl-4-Bora-3a,4a-Diaza-s-Indacene) (Invitrogen) diluted in 2% DMSO, then incubated for 1 hour at 28  °C in the dark. BODIPY C-5 specifically binds Short Chain Fatty Acids (SCFAs). Larvae were anaesthetized following2 . BODIPY C-5 was excited at 505 nm (blue light) and emitted a spectrum of wavelength light which peaked at 515 nm. Regions of Interest (ROI) of 15 gallbladders and 15 intestines per group were selected. Fluorescence intensity (a.u.) was determined using Image J software64 .

Lipid analogues and/or reporters were dissolved in DMSO to prepare stock solutions at the following concentrations: 5 mM (PED6, Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and 6.4 mM (BODIPY-C5, BODIPY-C16; both Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA). Following the manufacturer’s instructions, EnzChek (Invitrogen) was dissolved in 1× digestion buffer to prepare a 400 µg/mL stock solution. All the solutions were aliquoted in 20 µL volumes and stored at −20 °C.