Ems3 sycop3

Transgenic larvae were pre-screened for fluorescence using a zoomscope (EMS3/SyCoP3; Zeiss; Plan-NeoFluor Z objective). For multi-day imaging experiments, larvae were anesthetized and mounted in a Z-wedgi device [43 (link),48 (link)] where they were oriented such that the hindbrain was fully visible. Z-series images (5 μm slices) of the hindbrain were acquired on a spinning disk confocal microscope (CSU-X; Yokogawa) with a confocal scan head on a Zeiss Observer Z.1 inverted microscope, Plan-Apochromat NA 0.8/20x objective, and a Photometrics Evolve EMCCD camera. Between imaging sessions larvae were kept in E3-MB with PTU in individual wells of 24- or 48-well plates. Neutrophil-fungal interactions were imaged using an inverted epifluorescence microscope (Nikon Eclipse TE3000) with a Nikon Plan Fluor 20x/0.50 objective, motorized stage (Ludl Electronic Products) and Prime BSI Express camera (Teledyne Photometrics). Environmental controls were set to 37°C with 5% CO₂. Images were acquired every 3 min for 12 h. Imaging of A. fumigatus stained with CFW was performed using an upright Zeiss Imager.Z2 LSM 800 laser scanning confocal microscope with Airyscan detection and a Plan-Apochromat 20x /0.8 objective. A single z plane image was acquired for each hypha. Images were captured using identical laser and exposure settings for each condition.

At 2 dpf zebrafish were placed into a 60 mm dish in E3 +0.2 mg/ml Tricaine. A fine tip cautery (BVI Accu-Temp; cat #8442000) was used to burn the caudal fin. The cautery was turned on after being placed into the water and was held on the posterior tip of the caudal fin for 1–2 s, until a slight bend in the fin was apparent. After the burn, fish were placed into a clean milk-treated 35 mm petri dish with fresh E3 and held in an incubator at 28.5 degrees. For regeneration assessment, embryos were imaged at 1 and 2 dpw on a Zoomscope (EMS3/SyCoP3; Zeiss; Plan-NeoFluar Z objective). Images were taken on an Axiocam MRm CCD camera using ZEN pro 2012 software (Zeiss).

C3a.1^+/− adults were in-crossed. 3 dpf larvae were wounded by tail transection using a no. 10 Feather surgical blade. To visualize neutrophils in the wound microenvironment, the larvae were fixed at 2 hpw or 8 hpw in 4% paraformaldehyde in 1X PBS overnight at 4 °C. Sudan Black B staining was performed as described previously^{64 (link)}. Fixed larvae were imaged using a zoomscope (EMS3/SyCoP3; Zeiss; Plan-NeoFluar Z objective) and then genotyped as above. For macrophage quantification, c3a.1^+/− adults carrying a mpeg1:GFP transgene^{18 (link)} were in-crossed. At 3 dpf, larvae were pre-screened for fluorescence on a zoomscope. Tail wounding was then performed as described above and the larvae were fixed in 1.5% formaldehyde overnight at 4 °C. Fixed larvae were imaged using a zoomscope and genotyped as above. All image analysis was performed using Zen 2012 (blue edition, Carl Zeiss), blinded to genotype.