Calcein

The calcium tracker calcein [10 mg/kg body weight (BW), Sigma Aldrich, U.S. No. C087-5G] alone or both calcein and tetracycline (30 mg/kg BW, Sigma Aldrich, U.S. No. T3383) were injected to 3-month-old female rats (n = 8) on days 8 and 7 before mating. Four rats were injected with calcein (once a day for 2 consecutive days) and four other rats were injected with calcein and tetracycline (once a day for 2 consecutive days). After confirmed mating with vaginal swab smear test, these rats were then permitted to gestation for 21 days followed by euthanasia to isolate their fetuses. Then we randomly selected two pups from each litter for sectioning. Fetal tibias were embedded into paraffin while longitudinal and transverse sections were examined under a Zeiss 1500 fluorescence microscope. We adopted the 555 nm excitation wavelength and collected signal at 603 nm emission wavelength for tetracycline (Macri-Pellizzeri et al., 2018 (link)); therefore, it appeared as red as shown in our figures. calcein exhibited green fluorescence under emission at 514 nm and excitation at 475 nm wavelengths.

Calcein labeling histomorphometric analysis was performed as reported previously.^{22 (link)} Mice were intraperitoneally injected with Calcein (Sigma-Aldrich, USA) at 15 mg·kg⁻¹ prepared in 2% sodium bicarbonate solution at 10 days and 3 days before sacrifice. Bone formation analyses using MAR and BFR were performed according to the standardized nomenclature for bone histomorphometry under a fluorescence microscope (IX71; Olympus, Japan).

For preparation of CPPs 200 µl RPMI1640/10% FBS (room temperature) were mixed with 5 µl 100 mM CaCl₂ (2.5 mM [Ca²⁺]) in a 1.5 ml Eppendorf tube, vortexed and centrifuged for 2 h at 16,000 × g at 21 °C. For calcein-stained CPPs, “CPP-medium” was supplemented with 15 µM calcein (Sigma). After pelleting of spontaneously formed CPPs for 2 h, supernatant was carefully removed. For stimulation with CPPs, the CPP pellet was resuspended with 1 mM [P_i] containing cell culture medium to prevent the formation of new CPPs. 1x CPPs describes the CPP content out of 200 µl “CPP-medium” from one 1.5 ml tube, 2x CPPs describes the CPP amount out of two tubes. The 2x CPP concentration was used for stimulation, if not indicated otherwise. For detection of fetuin-A in CPPs, the CPP pellet (3x CPP) was washed in FBS-free 1 mM [P_i] RPMI1640 and again centrifuged for 2 h before resuspension in Laemmli buffer (reducing conditions), and loading onto a 10% acrylamide-SDS-gel and transfer to a PVDF membrane (GE Healthcare) via wetblot. Fetuin-A was detected with the goat-anti-Fetuin-A antibody (N-20, Santa Cruz). CPP medium was filtrated with a Whatman^® Anotop^® sterile syringe filter 0.1 µm.

At the appropriate time point, larvae were stained with a 0.2% calcein (Sigma) solution in fish water. A subset of fish were also stained with a 0.05% alizarin red (Sigma) solution in fish water 3 days prior to calcein staining. For imaging, stained zebrafish larvae were anesthetized in 0.01% MS-222 (Sigma) and mounted into borosilicate glass capillaries using 0.75% low melt-agarose (Bio-Rad) diluted in system water containing 0.01% MS-222. Capillaries were set on a custom 3-D printed holder to aid manipulation and rapid orientation of the specimen. Three-channel (GFP, DsRED, DAPI) images were collected on a high-content fluorescent microscopy system (Zeiss Axio Imager M2, constant exposure settings for all experiments) using a 2.5x objective (EC Plan-Neofluar 2.5x/0075). For each fish, a composite image stack (usually 3/1 images in the x/y directions; and optimized to 30-70 μm slice intervals in the z direction across the entire region of interest, usually about 9 slices; all at 2.58 μm/pixel) was acquired in mediolateral and anteroposterior views. Maximum intensity projections were generated from image stacks in Fiji for analysis. Following imaging, fish were collected for genomic DNA extraction and genotyping.