Plan fluor 10 0.30 dic l n1

For chemotaxis assays, migration of explants to Sdf-1 was assessed using a bead assay (Theveneau et al., 2010 (link)). This was done by incubating heparin-acrylic beads (Sigma-Aldrich) overnight at 4°C in PBS supplemented with 1 µg/ml Sdf-1 and placing the beads ∼1 mm apart in a line of silicone grease (VWR) on fibronectin-coated dishes. Explants were then plated perpendicularly at a distance of 250–500 µm. To test the effects of Ca²⁺ buffering, explants were incubated for 30 min with 50 µM BAPTA-AM (Cambridge Bioscience) or EGTA-AM (AnaSpec). Time-lapse imaging was performed in Danilchick’s medium using an upright microscope (Eclipse 80i; Nikon) fitted with an objective (Plan Fluor 10×/0.30 DIC L/N1) and a camera (ORCA-05G; Hamamatsu Photonics). Data were acquired using SimplePCI software. Tracking of migrating neural crest cells was performed using the ImageJ Manual Tracking plug-in. Immunocytochemistry was performed using a primary rabbit antibody to phosphopaxillin Tyr118 (1:200 dilution; EMD Millipore; Theveneau et al., 2013 (link)). Explants were costained with 2 µg/ml phallodin and 2 µg/ml DAPI.

For time-lapse recordings, images were captured every 3–5 min for a total of 8 h using Plan Fluor 10×/0.30 DIC L/N1 objectives with DM5500 and DMRXA2 compound microscopes (Leica Biosystems) at 18°C with either a DFC 300FX camera (Leica Biosystems) and LAS acquisition software or an Orca-5G camera (Hamamatsu Photonics) and SimplePCI software. For in vivo imaging, embryos were immobilized onto plasticine. Time-lapse and NCC tracking was performed using the ImageJ Manual Tracking plug-in as previously described (Carmona-Fontaine et al., 2008 (link); Matthews et al., 2008 (link)). Time-lapse imaging for CIL and CoA assays was performed at 18°C in Danilchick’s medium using an upright microscope (Eclipse 80i; Nikon) fitted with an objective (Plan Fluor 10×/0.30 DIC L/N1) and a camera (ORCA-05G; Hamamatsu Photonics). Data were acquired using SimplePCI software. Confocal images were acquired at 22°C in Danilchick’s medium using a TCS SPE upright microscope (Leica Biosystems) fitted with a HC PL APO 20×/0.75 IMM CS2 water objective. ISH images were captured at 18°C using a stereomicroscope (MZ FLIII; Leica Biosystems) fitted with a Plan 1.0×/0.125 objective and a camera (DFC420; Leica Biosystems). Data were acquired using IM50 v5 software (Leica Biosystems).