Larval zebrafish were live imaged after anesthetising them with MS222 tricaine and mounting them on glass coverslips, embedded in 1.5% low melting point agarose (Invitrogen). Z-stacks were acquired at relevant locations along the spinal cord using the LSM 880 confocal microscope equipped with Airyscan Fast and a 20X objective (Zeiss Plan-Apochromat 20X dry, NA=0.8). Z-stacks were acquired with an optimal z-step according to the experiment.
To follow the fate of oligodendrocytes, and myelination profiles over time, zebrafish were repeat imaged (4dpf pre-treatment, 3hpt, 1dpt, 3dpt, 5dpt and 7dpt). To do so zebrafish were carefully cut out from agarose following imaging and returned to E3 embryo medium, whilst being checked for signs of impaired health or swim behaviour. Zebrafish were maintained at 28.5°C in 10mM HEPES-buffered E3 embryo medium between imaging sessions.
To assess any developmental effects of the Tg(mbp:TRPV1-tagRFPt) transgene zebrafish were anesthetised with MS222 tricaine and imaged using the Vertebrate Automated Screening Technology BioImager system (Union Biometrica), which automated the positioning of larvae and image acquisition using a Zeiss spinning disk confocal microscope (VAST-SDCM)30
.
To follow the fate of oligodendrocytes, and myelination profiles over time, zebrafish were repeat imaged (4dpf pre-treatment, 3hpt, 1dpt, 3dpt, 5dpt and 7dpt). To do so zebrafish were carefully cut out from agarose following imaging and returned to E3 embryo medium, whilst being checked for signs of impaired health or swim behaviour. Zebrafish were maintained at 28.5°C in 10mM HEPES-buffered E3 embryo medium between imaging sessions.
To assess any developmental effects of the Tg(mbp:TRPV1-tagRFPt) transgene zebrafish were anesthetised with MS222 tricaine and imaged using the Vertebrate Automated Screening Technology BioImager system (Union Biometrica), which automated the positioning of larvae and image acquisition using a Zeiss spinning disk confocal microscope (VAST-SDCM)30
.