Calcein green

For fixed samples, all specimens (at 10 dpf and 30 dpf and three month old fish) were euthanized with an overdose of MS 222 and subsequently fixed for 12 h in neutral buffered 4 % paraformaldehyde. All specimens were stained for 15 min with 0.01 % ARS (3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt, from Sigma-Aldrich, St. Louis, MO) dissolved in 70 % ethanol [40 (link)]. For a better visualization of the mineralized structures in adult fish, specimens were macerated with 3 % KOH for 12 h and subsequently dissected.
For vital staining, three ARS concentrations (0.005, 0.01 and 0.05 %) were prepared in embryo medium [3 ]. The pH was adjusted to 7.4 with KOH. No precipitated ARS occurred in any of the three concentrations.
For the study of bone development, the specimens were transferred with a minimum volume of embryo medium to a new 24-well plate [3 ] with 3 ml of staining solution or new embryo medium (control). The animals remained in the staining solution for 15 min. Staining was performed once a day from 6 to 10 dpf, in each of the three ARS solutions described above. 0.2 % calcein [28 (link)] was used as a reference dye for mineral staining. In this case, larvae were stained for 10 min, as previously described [28 (link)]. Following staining with ARS, larvae were rinsed in embryo medium 3 times for 5 min, while larvae stained with calcein had to be rinsed at least 3 times for 10 min with embryo medium. In all cases, we assured that no dye residues were externally visible after the last rinsing period. If so, additional rinsing was conducted.
Stress levels were assessed by observing variations in the opercular movement frequency, as previously described [34 (link)], upon fish immersion during the first minute of staining and for 1 min at end of the staining period, before rinsing. The remaining period (remaining staining periods, and washing steps) prior to skeletal tissue imaging, took place in a dark environment to avoid stress. However, our personal observations suggest that there is no apparent effect on staining efficiency or fish health if animals remain exposed to light.
For regeneration studies, 5 adult specimens (3 month old) were exposed for 15 min to 0.01 % ARS solution prepared in system water prior to amputation and every 24 h thereafter, until 96 h post amputation (hpa). Adult fish were rinsed 3 times after each staining event for 5 min also in system water.
After ARS and calcein staining, larvae and adult fish were kept for periods no longer than 30 min prior to imaging. All specimens were anaesthetised up to 0.6 mM Tricaine solution (MS222; Sigma, St. Louis, MO) prior to microscopy analysis. Imaging was performed under green (510–550 nm) and blue (450–480 nm) fluorescent light to image ARS and calcein staining, respectively, and under visible light for total length (TL) measurements. Images were captured using a Leica MZ6 stereo microscope (Leica Microsystems, Germany) equipped for epifluorescence together with a F-View II camera, and Cell^Fv2.7 software (Olympus Soft Imaging Solutions GmbH, Germany). Higher magnifications of skeletal structures were visualised using an Axio Imager Z2 microscope equipped with a digital AxioCam ICc3 camera (Zeiss, Germany).
Tg(fli1:egfp) transgenic fish [51 (link)] were used to validate the suitability of ARS vital staining applied to GFP labelled fish during the regeneration of the caudal fin rays and the development of caudal vertebrae.
ARS staining was also used to detect skeletal deformities. The analysed deformities were not induced, but developed under regular rearing conditions. All fish were photographed using the equipment and the procedures described above.

To visualize viable cells by fluorescence microscopy staining with acetoxymethyl precursor of calcein (ViaStain Calcein AM, Nexcelom Bioscience) was performed. Calcein, a dye capable of permeating cell membranes, can be introduced into cells through incubation. Following the entry into the cells, endogenous esterase hydrolyzes calcein, transforming it into the intensely negatively charged green fluorescent calcein, which is exclusively retained in the cytoplasm of viable cells. 4× staining solution prepared in PBS was added to the culture media of wells containing cells or organoids and incubated for 30 min in the dark at 37 °C.

Following surgery, sheep were recovered and resumed weightbearing activity. Sheep were closely monitored twice daily for any signs of pain or distress. Sheep displaying such signs were promptly treated with Buprenex (0.005–0.01 mg/kg) and/or additional fentanyl patches. Each sheep received injections of calcein green (10 mg/kg) on two separate occasions. Solution preparation was described previously [19 (link)]. The first injection was administered intravenously 16 days before euthanasia, while the second occurred 5 days prior to euthanasia. These two injections of calcein green allowed for the assessment of bone viability and determined the rate at which bone re/modeled. HO has been shown to predominantly occur within 3 to 12 weeks after injury; however, it can take up to 6 months to manifest [24 ]. Sheep were humanely euthanized 24 weeks post-surgery with an injection of Beuthanasia D (1 mL/4.5 kg). A total of n = 35 of the sheep from the 7 study groups reached their pre-determined endpoint, while n = 1 was euthanized early due to a broken leg that was unrelated to the surgery (Table 1).