Eclipse 90i upright microscope

For live imaging, embryos were anesthetized in 0.016% tricaine (MS‐222; Sigma‐Aldrich) and embedded in 1.2% low melting agarose (Bio‐Rad, USA) in 35 mm glass‐bottom dishes (Eppendorf, Hamburg, Germany), and covered with 2‐mL fish medium containing tricaine. A Nikon SMZ18 epi‐fluorescence microscope was used for capturing whole‐embryo images. Z‐stack images and time‐lapse movies were recorded using a Nikon FV3000 confocal microscope (Nikon, Minato City, Tokyo, Japan) equipped with a ×30/1.05, WD 0.8 silicon oil objective. Time‐lapse files were processed using Imaris (Bitplane AG, Zurich, Switzerland) and Fiji (ImageJ; NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/) software to generate movies. Z‐stack images were processed into maximum intensity projections and channels merged using Fiji. Alizarin Red‐stained bone samples were imaged using a Nikon Eclipse 90i upright microscope equipped with NIS‐Elements version BR 3.0.