Determination of the proper ARS concentration for vital staining. Imaging of 6 dpf stained larvae with the same settings showed that (a) 0.05 % ARS 15 min immersion yielded stronger staining than (b) 0.01 % ARS, but the later provided the best signal to noise ratio, with minimum stress levels. c 0.005 % ARS was considered the lowest concentration providing signal detection, since most structures were weakly stained. d 0.2 % calcein staining was used as a reference staining. e Graphical representation of mineral apposition rates (columns) at 24, 48 and 72 h after first staining, when exposed to 0.005, 0.01 and 0.05 % ARS and 0.2 % calcein. Bars represent standard deviation. Means were statistically different (*p < 0.05), by multiple comparison of means using one-way ANOVA and Tukey?s post test, between larvae stained with calcein and those stained with 0.005 % (0.29 % less apposition rate with calcein, 82 % of the 0.005 % ARS value) and 0.01 % (0.26 % less mineral apposition rate with calcein, 83 % of the 0.01 % ARS value) ARS at 24 hps. On the second axis of the graph, growth (inferred by increase in TL) is indicated: control conditions (black dots; n = 17); following staining with 0.005, 0.01 and 0.05 % ARS, and 0.2 % calcein (white dots; n = 17). Scale bars = 1 mm

Sequence of lepidotrichia regeneration events in the zebrafish caudal fin. Caudal fin of fish stained with 0.01 % ARS at a 24, b 48, c 72 and d 96 hpa. b′ Detail of a fin ray at 48 hpa, already displaying de novo mineralized tissue. Amputation axis is indicated (dashed line). Scale bar (a-d) = 2 mm; (b′) = 0.2 mm

ARS staining of fixed zebrafish samples. Panels a-b show a vertebral column of a 10 dpf larva stained with 0.01 % ARS in 70 % ethanol. a Bright field observation provides less detail of the early mineralization deposits than b fluorescence observation (arrowheads). Panels c-d show cranial structures of a juvenile (30 dpf, 8 mm TL) stained with 0.01 % ARS and observed under c bright field and d fluorescent light, evidencing the higher power of detection of, e.g., the operculum (arrowheads) under fluorescent conditions. Scale bars (a, b) = 0.04 mm; (c, d) = 0.2 mm

ARS fluorescence sensitivity single or in combination with expression of green fluorescent reporters. Macerated abdominal vertebrae of adult fish in a sagittal view and b transverse view show distinct mineralization fronts, indicative of vertebral growth. c Caudal fin ray of an adult Tg(fli1:eGFP) fish stained with 0.01 % ARS and d caudal vertebrae formation in a Tg(fli1:eGFP) zebrafish larva. There is a clear distinction between structures stained with ARS and structures that express GFP. Scale bars (a, b) = 0.1 mm; (c, d) = 0.2 mm

Detection of skeletal malformations in zebrafish. Deformed bony structures in a caudal fin rays and b-c different regions of the vertebral column. All regions display affected structures with different degrees of severity. White arrowheads show sites of malformation. Scale bars (a) = 2 mm; (b-c) = 0.4 mm