FIGURE SUMMARY
Title

Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Authors
Bensimon-Brito, A., Cardeira, J., Dionísio, G., Huysseune, A., Cancela, M.L., Witten, P.E.
Source
Full text @ BMC Dev. Biol.

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

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ BMC Dev. Biol.