Visualizing Calcium Dynamics and Macrophage Activation in Live Zebrafish Larvae

Larvae were anesthetized with 100 μg/mL buffered tricaine and mounted in 1% low-melting point agarose as previously described (41 (link)). Epi-fluorescence microscopy was performed using a MVX10 Olympus microscope (MVPLAPO 1X objective; XC50 camera). Confocal microscopy was performed on ZEISS LSM880 FastAiryscan, using 20X/0.8 objective, plan apochromat equipped with DIC for transmission images, resolution at 512x512 pixels. The wavelength were respectively 488nm (Argon Laser) and 561nm (DSSP Laser) for excitation. Detection was selected at 505-550nm for PMT detector and 585-620nm for GaAsP detector. The images were taken in a sequential mode by line. The 3D files generated by multi-scan acquisitions were processed by Image J. To image Ca²⁺ oscillations at the wound, we used ANDOR CSU-W1 confocal spinning disk on an inverted NIKON microscope (Ti Eclipse) with ANDOR Neo sCMOS camera (20x air/NA 0.75 objective). Image stacks for time-lapse movies were acquired at 28°C every 20 seconds (s), with z-stack of 45 μm at 3 μm intervals. To image macrophage activation in live, z-stacks of 78 μm with 3 μm intervals were acquired every 3min, in multiposition mode. The 4D files generated from time-lapse acquisitions were processed using Image J. Brightness and contrast were adjusted for maximal visibility.