HCR fluorescent in situ hybridization (FISH)

HCR RNA in situ hybridization (Molecular Instruments) experiments on 14 µm cryosections of fixed 3 and 5 dpf larvae and adult brains were performed as previously described (Choi et al., 2018). Briefly, sections were air dried and re-fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Following fixation, sections were further permeabilized using 1 µg/ml Proteinase K (ThermoFisher) for 5 min, followed by PBS washes. Slides were then put through an ethanol dehydration series of 50%, 70% and two rounds of 100% ethanol and air dried for 5 min. Dried slides were put into pre-hyb solution for at least 10 min at 37°C. Subsequently, the probes for mfsd2aa, mfsd2ab, slc2a1a and mouse Gfap were added to the slides at a final concentration of 4 nM to hybridize overnight at 37°C. The next day, slides were washed with a series of wash buffer to 5x SSCT (5x SSC with 0.1% Tween 20). Excess liquid was then removed and samples were immersed in amplification buffer at room temperature for 30 min prior to hairpin amplification, which occurred overnight at room temperature. The next day, slides were washed in 5x SSCT and mounted with Fluoromount-G (Electron Microscopy Sciences).