Voltalef oil

Fixed preparations were imaged using an Olympus IX81 (40×/1.3UPlan FLN Oil or 60×/1.35 UPlanSApo Oil). Live imaging was performed using a Leica SP8 (63×/1.4 HCX PL Apo CS Oil) or Olympus IX81 (40×/1.3 UPlan FLNOil or 60×/1.35 UPlanSApo Oil) inverted confocal microscope. For live observations, ovaries were dissected and imaged in 10S Voltalef oil (VWR Chemicals).

Embryos were laid overnight at 23°C on apple juice agar dishes, dechorinated in bleach and mounted on Greiner Lumox gas-permeable culture dishes (Sigma) in halocarbon oil 700 (Sigma) or on glass slides with double-sided tape with Voltalef oil (VWR International). For experiments using RNAi, embryos were laid at 28°C. Embryos were wounded at stage 14 using a nitrogen ablation laser (Spectra-Physics) attached to a Zeiss Axioplan 2 widefield imaging system. Confocal imaging was performed on a Leica SP5-II confocal laser scanning microscope. Image preparation and analysis was performed on maximum projected confocal movies and utilised Volocity (PerkinElmer) and ImageJ (NIH) software. Graph plotting and statistical analysis were performed using Prism (Graphpad). Image quantification methods can be found in the supplementary material.

Embryos of the appropriate developmental stage were collected from overnight apple juice plates, dechorionated in bleach for 1 min and mounted on double-sided sticky tape on glass slides in 10S Voltalef oil (VWR). Wounds were induced using a nitrogen-pumped Micropoint ablation laser tuned to 435 nm (Andor Technologies) (Razzell et al., 2013 (link)). Microinjections and UV-induced apoptosis were performed as before (Tan et al., 2014 (link), Moreira et al., 2010 (link)). For Amplex Red staining, dechorionated embryos were incubated in a 1:1 mixture of heptane:Amplex Ultrared solution (50 μM in PBS) for 30 min and mounted as above. Imaging was performed on a PerkinElmer UltraView spinning disc system or Leica TCS SP5 confocal microscope. Image processing was performed using ImageJ (NIH), Adobe Photoshop, or Adobe Illustrator software. For a detailed description of image processing and analysis, see Supplemental Experimental Procedures.

Developmental stage 15 embryos were collected in cell strainers (Falcon), dechorionated with bleach (Jangro), washed vigorously with water and mounted between a glass slide and a supported coverslip in droplets of VOLTALEF oil (VWR) as previously described (Evans et al., 2010 (link)). Ventral hemocytes were then imaged using a spinning disc confocal microscope (Perkin Elmer Ultraview) with a plan-apochromat 63× objective with a NA of 1.4 and a Hamamatsu C9100-14 camera. The acquisition software used was Volocity (Perkin Elmer). Epithelial wounds were generated using laser ablation (nitrogen-pumped micropoint ablation laser tuned to 435 nm, Andor Technologies) as previously described (Wood et al., 2002 (link)).

Embryos were washed off apple juice agar plates, dechorionated in bleach, and then washed in distilled water. Live and fixed embryos were mounted on slides in voltalef oil (VWR) or DABCO (Sigma), respectively, as per Evans and colleagues, 2010 [9 (link)]. Immunostained embryos were imaged on a Nikon A1 confocal system using a 40X objective lens (CFI Super Plan Fluor ELWD 40x, NA 0.6), which was used for the migration movies in Fig 4. Aside from S2 Movie (Zeiss Lightsheet system; see S1 Methods), all other timelapse imaging and wounding was performed on a Perkin Elmer UltraView Spinning Disk system using a 40X objective lens (UplanSApo 40x oil, NA 1.3). Lower magnification images of embryos were taken using a MZ205 FA fluorescent dissection microscope with a PLANAPO 2X objective lens (Leica) or a 20x objective (UplanSApo 20x, NA 0.8) on the Perkin Elmer UltraView Spinning Disk system.

Embryos laid on apple juice/agar plates were washed off into a cell strainer and dechorionated in 5% bleach for 1 min, followed by five washes in distilled water. Embryos were mounted in 10S Voltalef oil (VWR) as per Evans et al., 2010b (link). Live embryos were imaged using a Perkin Elmer Ultraview Spinning disk system using either a 10× air (UplanSApo 10×/NA 0.4; lateral images of stage 15 embryos to show developmental dispersal of macrophages or to quantify total number of macrophages per embryo) or 40× oil immersion (UplanSApo 40× oil/NA 1.3; all remaining live imaging) objective lens. For analysis of macrophage random migration and wound responses, the ventral surface of stage 15 embryos was imaged to a depth of approximately 20 μm with a 1 μm spacing between z-planes. Time-lapse movies were assembled from z-stacks taken every 2 min for 1 h using Volocity software (Perkin Elmer) for analysis of both macrophage random migration and wound responses. Wounding was performed using a Micropoint ablation laser (Andor) to ablate the ventral epithelium on the ventral midline in the medial-most segments of the embryo as per Evans et al. (2015) (link); the inflammatory responses of macrophages in this region were then followed for 1-h post wounding.