Alcian Blue staining

Stage 40 (9 dpf at 26°C) medaka hatchlings were anaesthetized with 1× tricaine, and the tail was clipped and processed for PCR genotyping. Specimens were fixed in 4% paraformaldehyde in PBS overnight at 4°C. Samples were dehydrated in 50% and 70% ethanol for 15 min at room temperature (RT). Cartilage was stained in 0.02% Alcian Blue 8GX (Sigma-Aldrich) in 70% ethanol and 60 mM MgCl₂ overnight at RT. Hatchlings were washed once in 70% and 50% ethanol and eye pigments were bleached in 1% KOH, 3% H₂O₂ in PBS for 30 min at RT. Hatchlings were washed once with 50% ethanol and imaged under Nikon SMZ18 in 3% methylcellulose. For long-term storage, samples were dehydrated in 100% ethanol and kept at −20°C. Using Fiji (Schindelin et al., 2012), analysis of cartilage lengths (Euclidean distance) was performed according to Cline et al. (2012), i.e. individual cartilage lengths were normalized to the standard length (SL) which is the distance between the lenses of the eyes. Two-tailed nonparametric Student's t-test was applied for statistics.