Hybridization Chain Reaction (HCR) FISH staining

HCR probes, hairpins, and buffers were purchased from Molecular Instruments. Staining was performed using the manufacturer’s protocol: “HCR RNA-FISH protocol for whole-mount zebrafish embryos and larvae (Danio rerio)” with the following modifications: we did not apply PTU to inhibit melanogenesis, we used 30 larvae per Eppendorf tube, and we used 8uL of 1uM cdh16 probe solution instead of 2ul as suggested in the protocol. Animals were mounted laterally in 1.5% low-melt agarose in PBS and imaged using a 63x objective on a 3i Marianas Spinning Disk Confocal Microscope.