2.6. Alcian Blue Staining

Alcian blue 8 GX (Sigma-Aldrich) was used to visualise cartilage glycosaminoglycans (GAGs). Larvae (5 dpf) were fixed in phosphate buffered formaldehyde (3.5% formaldehyde diluted in 100 mM monosodium phosphate buffer supplemented with 25 mM disodium phosphate) for an hour. Following fixation, larvae were dehydrated to 80% ethanol (EtOH). Larvae were incubated in freshly made Alcian blue staining solution (0.02% Alcian blue in 70% ethanol supplemented with 80 mM MgCl₂) at room temperature for 1 h. Larvae were washed in 50% EtOH, and subsequently washed in deionised water with 0.2% Triton-X100. Pigmentation was bleached in a solution of 10% KOH, 1.5% H₂O₂, in deionised H₂O) which was followed by a wash in 10% KOH, 10% Glycerol, and cleared in 70% glycerol, prior to imaging (ventral and lateral views) with a Leica stereomicroscope.