2.6. Alcian Blue Staining

Alcian blue staining of cartilages was carried out as described previously with minor modifications [9,42]. Briefly, zebrafish larvae were collected at 96 hpf, fixed in 4% PFA overnight, and treated with alcian blue solution (0.1%) dissolved in 80% ethanol/20% glacial acetic acid overnight. Specimens were subjected to several washes of acid alcohol and then transferred into 1% potassium hydroxide/3% hydrogen peroxide solution to further clear pigmentation. Subsequently, embryonic tissues were digested in 0.05% trypsin for two hours. Cartilage preparations were mounted in 70% glycerol and visualized on a SteReo Lumar V12 microscope from Carl Zeiss.