Calcein powder

Calcein solution (0.2%) was prepared by dissolving 2 g of calcein powder (Sigma, Life Sciences) in 1 liter of deionized water. Due to calcein's strong acidifying effects, an appropriate amount of Sodium hydroxide (NaOH; 0.5 N) was added to the solution to have the pH 7.4. Treated zebrafish embryos were netted and immersed in the solutions in Petri dishes for 10 min. After the immersions, the embryos were rinsed in fresh water for 10 min in order to eliminate the excess of calcein. The embryos were then euthanized in 3% solution of tricaine-methanesulfonate (MS222) and mounted on glass slides. Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). Observations were carried out using confocal laser scanning microcopy (CLSM; Zeiss LSM 700), equipped with the ZEN-2011 software.

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.