Vital Staining of Mineralized Bone in Zebrafish

For most observations we vitally stained mineralized bone in E2 embryo medium containing 50 µg/ml Alizarin Red S (JT Baker catalogue # A475-03) and 10 mM HEPES, pH 7.0. Larvae were stained for 1 to 2 hours in the dark and juveniles (after 21 dpf) were stained overnight in the dark. Fish were rinsed well and for mounting were anesthetized in E2 with 0.017% Tricaine (3-amino benzoic acid ethyl ester, Finquel, from Argent Cat# C-FINQ-UE-5G). The fish were mounted in 0.2% agarose in E2 medium (Ultra-Low gel temperature Type IX Agarose, SIGMA catalogue number A5030) on a drop of 0.3% methyl cellulose in E2 medium between bridged cover slips [47] . After the agarose gelled, the cover slip mount was flooded with the tricaine solution. We imaged preparations with a Zeiss LSM 5 Pascal confocal scanning microscope with AIM software, using a 543 nm excitation laser. We crafted scan settings with attention to capturing the entire opercle in x, y and z planes. To increase resolution, we used a slow scan speed, a very small z slice interval and a small pinhole optimized to 1 airy unit. We used an averaging of 2 to 3 slices. We present image stacks as projections, saved as TIFF files for morphometric analyses.
To observe bone growth between two stages, we labeled larvae with successive pulses of, first, 50 µg/ml Alizarin Red S, and after a period of wash out, a second pulse of 50 µg/ml Calcein (*high purity*, Molecular Probes catalogue # C481, in E2 embryo medium buffered with 1 mM Sodium Phosphate to pH 8.0). We report pulse times in the Figure 4 legend for the individual experiments. We rinsed the fish well, and imaged as above using the 543 nm and the 488 nm lasers and multi track scanning. We used the same settings for two color imaging of Alizarin Red bone staining and eGFP transgenic cell labeling.
For the pulse labeling experiment with the young adult shown in Figure 4, the fish was vitally stained with Alizarin Red S at 100 µg/ml, overnight between 42 and 43 dpf. The stain was washed out and after an 11-day interval of growth in the absence of stain the fish was euthanized at 54 dpf. The Op was dissected out, soft tissue was gently scrubbed away with a wooden toothpick, and the Op was imaged with a Zeiss Axiophot microscope (20x objective).
For the adult bones shown in Figure 8, the zebrafish Op was stained with Alizarin Red S after dissection from a euthanized, unfixed 40 dpf fish and photographed with oblique transmitted illumination to reveal banding and other features of the matrix, including the two struts described in the text. The sucker Op was picked up by one of us on the shore of Chateaugay Lake in New York (USA), and photographed as a dried preparation with flat incidence illumination. The northern pike Op was dissected from a fresh-frozen head, and photographed as for the sucker but after only partial drying to better reveal the banding. The pike head was a gift from Jonathan Gustafson of the Minnesota (USA) Department of Natural Resources, who noted from the image shown in Figure 8 that the fish appeared to be about 5 years old. Hence for the pike the more prominent bands (which are subdivided by narrower bands) are approximately annual, rather than diurnal as we have estimated for zebrafish.