A1r s confocal laser scanning microscope

Whole-mount fin images were acquired using an AxioZoom stereo fluorescence microscope (Zeiss) at the time points indicated. For imaging Wnt responsive cells, Tg(7xTCFXla.Siam:EGFP)^ia4 (tcfsiam:EGFP) fish were used, and images were processed using the Zen software (Zeiss) (69 (link)). Whole-mount 2 dpa fin innervation images were acquired using a Nikon A1R-s confocal laser scanning microscope and processed using the NIS-Elements software (Nikon). The 7 dpa fin innervation images were captured using a Keyence BZ-X800 fluorescence microscope and analyzed using Keyence software. Image stitching was automatically processed using NIS-Elements or Keyence software during image acquisition. Maximum projections were obtained using 30 and 20 slices every 1 µm for 2 and 7 dpa fin innervation images, respectively. Further image processing was carried out manually using Photoshop or FIJI/ImageJ software. Regenerate fin lengths were measured and quantified by averaging fin rays 3, 4, and 5 from the dorsal and ventral lobes of the fin.
In situ hybridization on cryosections of 4% PFA–fixed fins was performed as previously described (70 (link)). To generate digoxigenin-labeled probes for scn8ab, we used the 3′ untranslated region of scn8ab cDNA, which does not show conservation with other scn genes (60 (link)). Primer sequences of scn8ab, fgf20a, and fn1b are listed in SI Appendix, Table S1.

Fixed samples were imaged on a Nikon A1-RS confocal laser scanning microscope using a ×60 magnification, 1.4-numerical-aperture (NA) oil objective, and 405-, 488-, 561-, and 647-nm laser excitation. General live-cell microscopy was performed on a spinning disk microscope equipped with a ×60 magnification, 1.4-NA oil objective, and 488-, 561-, and 647-nm laser excitation. Images were collected on an electron-multiplying charge-coupled device (EMCCD) camera. Physiological conditions were kept constant at 5% CO₂ and 37°C throughout the experiments.