Csx x1 scan head

Confocal imaging of live or stained zebrafish larvae was performed using an Innovative Imaging Innovations (3I) spinning-disk confocal microscope equipped with a Yokogawa CSX-X1 scan head. Super resolution confocal analyses were performed using a Zeiss LSM 980 with Airyscan 2 confocal microscope.
For live imaging, zebrafish larvae were anesthetized with 0.16 mg/ml Tricaine in embryo medium and mounted in 1.2% low-melting agarose on a cover slip with extra embryo medium sealed inside vacuum grease to prevent evaporation. Time-lapse Ca²⁺ imaging was performed on 6 dpf Tg[slc1a3b:myrGCaMP6s] larval spinal cord or hindbrain on a single z-plane at 0.5 second intervals for 5–10 minutes. For drug treatment experiments, DMSO (0.1%) or norepinephrine (100 μM, Sigma Aldrich) in embryo medium were used. For TTX injection experiments, 6 dpf Tg[slc1a3b:myrGCaMP6s] larvae were injected with 1 nl 0.5 mM TTX into the yolk, and 10 minutes were allowed to elapse to confirm the larvae were paralyzed. After 10 minutes and confirmation of paralysis, DMSO or NE was applied to the embryo medium, and larvae were incubated for 20–30 minutes before Ca²⁺ imaging as described above.