Multimodal Imaging Analysis of Cytoskeletal Proteins

Myh10 en face apical endfoot images, Myh9 3D reconstructions, and Myh9 LHX6/Laminin images were captured using an Andor Dragonfly Spinning Disk Confocal plus with 40X and 63X objectives. All other images were captured using a Zeiss Axio Observer Z.1 with Apotome for optical sectioning. 20X, 40X, and 63X objectives were used. For each experiment, 3 to 4 sections per embryo were imaged. Identical exposures, apotome phase images, and Z intervals were used. Cell counting was performed manually (FIJI cell counter) or automatically (QuPath). For QuPath quantification, the following parameters were used: requested pixel size = 0.1 μm, background radius = 5 μm, minimum area = 10 μm², maximum area = 200 μm², cell expansion = 2 μm, include cell nucleus and smooth boundaries were unselected. For quantification of cells touching the BM, cells colocalizing with the BM label (Laminin or Collagen) were counted manually. Cortical, SOX2, MZ, and Calretinin thickness measurements were taken using the line tool in ImageJ.