Lsm880 fast airyscan

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.

The DRGs, spinal cord, and sympathetic ganglia were cut at a thickness of 25 μm using a freezing microtome (Leica HM430, Wetzlar, Germany), and all consecutive sections were collected. Local ST25 tissue was cut into 20-μm thick sections (Wang et al., 2018 (link); Zhang et al., 2021 (link)). All sections were mounted on slides, sealed with a drop of an anti-fluorescence quenching sealer (Abcam, Cambridge, MA, USA), and observed under laser confocal (ZEISS LSM880 + Fast Airyscan, Germany) and inverted fluorescence microscope (ZEISS Axio Scope AI, Germany) microscopes.

Stacked brightfield and fluorescence overview images were collected with 10 × , 40 × or 63 × Plan-Apochromat objectives using an automated slide scanner microscope (Zeiss AxioScan Z1) or a fluorescence-phase contrast microscope (Keyence BZ 9000). Stacks were collected at 1–5 µm intervals, merged and post-processed using the corresponding software (ZEN 2.3 or BZ-Analyzer). Shading correction and white balance were carried out. High magnification fluorescence images were obtained with a 63 × Plan-Apochromat (1.2 numerical aperture) objective using a confocal laser scanning microscope (Zeiss LSM 510 Meta or LSM 880 fast Airyscan) with lasers excitation at 543 and 633 nm and emission was detected using a BP 565–615 and a BP 650–710 filter.

For lipid chase experiments, AML cells were labeled with 1 mM of the fluorescent fatty acid BODIPY 558/568 C₁₂ (RC₁₂Thermo Fisher Scientific) overnight in complete culture medium. After two washes, AML cells were seeded on coverslips, fixed, and stained for mitochondrial network with TOMM20 (Genetex, 133756) like for LC3B staining. For imaging of the mitochondrial network and RC₁₂ distribution, 6–8 µm Z-stacks at 0.18 µm were used. Fluorescence signals were analyzed using high resolution fluorescence microscopy. Images were taken with a Zeiss LSM 880 FAST Airyscan using a 63× Plan-Apochromat objective with 1.4 aperture under immersion oil. The Manders 2 (M2) coefficient, or fraction of RC₁₂ signal overlapping mitochondrial network, was determined from Z-stack projections of RC₁₂ and mitochondrial network using the Fiji JACoP plugin.

Whole larvae were fixed in paraformaldehyde at 4°C for 48 h, cryoprotected in 30% sucrose and mounted in O.C.T. medium (Sakura, Tissue-Tek, Alphen aan den Rijn, the Netherlands). Larvae were transversely sectioned in 10-μm-thick slices using a cryostat (Leica, Wetzlar, Germany) at −20°C and mounted on glass slides. Cryosections were blocked with a solution containing 0.1% phosphate buffered saline/Triton X-100 and 5% horse serum for 30 min at room temperature. They were subsequently incubated at 4°C overnight with the following primary antibodies: mouse anti-Rho4d2 (1:7,000; ab98887, Abcam, Cambridge, UK) and mouse anti-Zpr-1 (1:500; ab174435, Abcam). After several washes, sections were incubated with specific secondary antibodies: Cy3 conjugated anti-mouse antibody (1:800; 715-165-150, Jackson ImmunoResearch, West Grove, PA), Cy3-conjugated anti-rabbit antibody (1:1,000; 711-166-152, Jackson ImmunoResearch), or Alexa Fluor 488 conjugated anti-mouse antibody (1:1,000; 715-545-150, Jackson ImmunoResearch). Nuclei were counterstained with 40,6-diamidino-2 phenylendole (DAPI) (1:5,000; Sigma Aldrich). The emitted fluorescence was measured using a confocal microscope (LSM880 Fastairyscan, Carl Zeiss, Jena, Germany).

Whole larvae were fixed in paraformaldehyde at 4 °C for 48 h, cryoprotected in 30% sucrose and mounted in O.C.T.^TM medium (Sakura, Tissue-Tek, Alphen aan den Rijn, The Netherlands). They were transversely sectioned in 10-µm thick slices using a cryostat (Leica, Wetzlar, Germany) at −20 °C and mounted on a glass slide. Cryosections were blocked with a solution containing 0.1% PBS/Triton X-100 and 5% horse serum for 30 min at room temperature. They were subsequently incubated overnight at 4 °C with the following primary antibodies: mouse anti-Rho4d2 (1:7000; ab98887, Abcam, Cambridge, UK), mouse anti-ZPR-1 (1:500; ab174435, Abcam) or rabbit anti-cleaved caspase-3 (Asp175) (1:500, #9661, Cell Signaling Technology, Danvers, MA, USA). After several washes, sections were incubated with the following conjugated secondary antibodies: Cy3 anti-mouse (dilution 1:800; #715-165-150, Jackson ImmunoResearch Europe Ltd., Ely, UK), Cy3 anti-rabbit (1:1000; #711-166-152, Jackson ImmunoResearch) or Alexa Fluor 488 anti-mouse (1:1000; #715-545-150, Jackson ImmunoResearch). Nuclei were counterstained with 40,6-diamidino-2 phenylendole (DAPI; 1:5000; Sigma Aldrich). The emitted fluorescence was measured using a confocal microscope (LSM880 Fastairyscan, Zeiss, Germany).

HeLa cells were seeded on μ-slide 8-well coverslips (Ibidi) and transfected with NS1-GFP (100 ng per well) using FuGENE in accordance with the manufacturer’s instructions. The next day, the cell medium was removed and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The cells were blocked and immunostained with an antibody specific for MED25 (Atlas Antibodies; HPA068802) and Alexa Fluor-568 conjugated secondary antibody. Nuclei staining was performed using DAPI. Images were acquired using a Zeiss LSM 880 Fast Airyscan.