To visualize bone repair in live zebrafish, ARS and calcein green stain were used. ARS solution contained 74 µM Alizarin Powder (Sigma-Aldrich) and 5 mM Hepes dissolved in Danieau’s buffer. Calcein green staining solution contained 40 µM calcein powder (Sigma-Aldrich) dissolved in Danieau’s buffer and was pH calibrated to pH 7.4. Live fish were immersed in either ARS or calcein green for 2 h, then immersed in fresh system water for 15 min before imaging to clear excess stain from the fish.
Fluorescence intensity at fracture sites was measured using ImageJ; lines were drawn along the fracture callous length, and then the line width was expanded to encompass the whole fracture. The mean intensity was then measured across this area, before using the same region of interest to measure the mean intensity of adjacent, uninjured hemirays. Fracture intensity was then normalized against the healthy bone to account for differences in baseline expression caused by reporter integration number.
Fluorescence intensity at fracture sites was measured using ImageJ; lines were drawn along the fracture callous length, and then the line width was expanded to encompass the whole fracture. The mean intensity was then measured across this area, before using the same region of interest to measure the mean intensity of adjacent, uninjured hemirays. Fracture intensity was then normalized against the healthy bone to account for differences in baseline expression caused by reporter integration number.