Mvplapo 1x objective

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.

Caudal fin fold amputation was performed in 3 dpf larvae under anaesthesia with 0.016% Tricaine (MS222, Sigma) in zebrafish water using a sterile scalpel^{57 (link)}. For imaging, live embryos were anesthetized in 0.016% Tricaine, and positioned in 35 mm glass-bottom dishes (FluoroDish^TM, World Precision Instruments). They were mounted in 1% low melting point agarose (Sigma) with Tricaine. Light microscopy was performed using an MVX10 Olympus macroscope equipped with an MVPLAPO 1X objective and XC50 camera. Z-stacks series were obtained using an inverted confocal microscope Leica TCS SP5 (Leica Application Suite V3.2) and TCS SP8 (Leica Application Suite V3.5) equipped with an HCXPL APO 40x/1.25-0.75 oil and an HC PL APO 0.70 ∞ (infinity) 20x objective (Leica). The mCherry signal was excited with a 560 nm laser, and GFP with a 490 nm laser. Datasets were analysed using Fiji Software (ImageJ 1.52p)^{58 (link)}.