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Cartilage Alcian blue staining
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Cartilage was stained via a modified version of established protocols (Delbaere et al., 2019; Gebuijs et al., 2019). Larvae were fixed for 6 h in 4% paraformaldehyde. Specimens were bleached for optical clarity in 1% hydrogen peroxide, 1% potassium hydroxide and 0.2% Triton for 1 h. Staining with Alcian blue was performed for 3 h and this was followed by a clearing step in acidic ethanol (5% hydrochloric acid, 70% ethanol) for 10 min then transferred to pure glycerol for imaging.
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