Im50 v5

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).

In situ hybridization on Xenopus embryos was performed as previously described (Harland, 1991 (link)). In brief, embryos were fixed in MOPS/EGTA/magnesium sulfate/formaldehyde buffer, dehydrated in methanol, rehydrated with PBS–0.1% Tween, treated with proteinase K, and bleached with hydrogen peroxide. Embryos were postfixed in formaldehyde and preincubated in hybridization buffer before incubation with digoxigenin-labeled RNA probes to Twist, Snail2 (Linker et al., 2000 (link)), and CAX. The latter was generated from Xenopus IMAGE clone 6640627 (RefSeq accession no. NM_001095931.1), linearized using Pst1, and transcribed with T7 RNA polymerase. Staining was visualized using a sheep antidigoxigenin antibody coupled to alkaline phosphatase (1:3,000 dilution; Roche). Images were captured 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).