A3882

For bone matrix staining, medaka larvae were incubated in 0.1% alizarin-3-methyliminodiacetic acid (alizarin complexone, ALC; Sigma A3882) or 0.01% calcein (Sigma C0875) in fish medium at 30°C for 1.5 or 2.5 h (for larvae at 9-17 dpf). After incubation, larvae were rinsed in fish medium for 30 min to 1 h before being mounted for imaging. To ensure that all larvae were at the same developmental stage for BP treatment, ALC staining was conducted to count mineralized caudal fin rays. Staining was analyzed using tetramethylrhodamine (TRITC) and GFP filter settings. Larvae with four mineralized caudal fin rays (equivalent to 9 dpf) were selected for further analysis.

Fluorochrome labeling using calcein green (15 mg/kg; C0875, Sigma-Aldrich, Saint Louis Mo, USA) and alizarin complexone (30 mg/kg; A3882, Sigma-Aldrich, Saint Louis, MO, USA) was performed 10 days and 1 day before the sacrifice, respectively. The reagents were dissolved in sodium bicarbonate solution and administrated subcutaneously [15 (link)].

Live staining of mineralized bone matrix was done by immersing medaka hatchlings (12 to 18 dpf) in either Alizarin Complexone solution (ALC; 0.1% in fish medium, Sigma A3882) for two hours or in a Calcein solution (0.01% in fish medium; Sigma C0875) for one hour in the dark at room temperature (RT). Stained hatchlings were rinsed with fish medium three times (15 mins per rinse, at RT) and mounted in 1.5% low melting agarose on a glass bottom petri dish for live fluorescence imaging (488 nm laser/GFP filter for Calcein; 568 nm laser/mCherry filter for ALC).

Standard bone histomorphometric measurements were performed using OsteoMeasure software (OsteoMetrics, Decatur, GA, USA) following the guidelines of the American Society for Bone and Mineral Research. For static histomorphometry, femur cortical bone parameters were analyzed by PharmaTest Services, Ltd (Turku, Finland). Briefly, femurs were fixed in 4% paraformaldehyde, dehydrated in 70% ethanol and embedded in methyl methacrylate. After embedding, femurs were sectioned longitudinally and 4-µm-thick sections were stained with Masson-Goldner’s Trichrome. For dynamic histomorphometry, mice and rats were dosed with the fluorochromes calcein (10 mg·kg⁻¹, Sigma-Aldrich C0875), demeclocycline HCl (30 mg·kg⁻¹, Sigma-Aldrich 1170000), and alizarin complexone (20 mg·kg⁻¹, Sigma-Aldrich A3882) to label active bone-forming surfaces. Distal tibia analyses were performed halfway between the tibia-fibular junction and the distal end of the tibia. Bones were embedded in methacrylate and thick sections (~80 μm) prepared with a Leica SP1600 Saw Microtome (Buffalo Grove, IL, USA).^{52 (link)}

Mineral deposition was evaluated by intraperitoneal injections of calcein (Sigma # C0875; 2.5 mg/kg body weight) and alizarin complexone (Sigma # a3882; 7.5 mg/kg) into pregnant females and, postnatally, into cubs. Two mice were used for each injection regime. Prenatally harvested limbs were fixated overnight in 4% PFA/PBS, dehydrated to 100% ethanol, embedded in paraffin and sectioned at a thickness of 7 μm. Postnatally harvested limbs were fixated 24 h in 4% PFA/PBS and gradually dehydrated from 70% ethanol to 100% ethanol twice for 48 h each time. Then, samples were infiltrated and embedded in JB-4 Embedding Kit (Electron Microscopy Science #14270–00) and sectioned longitudinally at a thickness of 7 μm. Fluorescence was visualized by confocal microscopy.
Confocal imaging was performed using a Zeiss LSM 510 upright confocal microscope (Carl Zeiss, Jena, Germany) with an EC Plan-Neofluar 10x/0.3 objective, NA 1.0. Calcein fluorochrome was excited with a 488 nm argon laser and alizarin with 561 nm argon laser. Following imaging, all images of the same section were stitched using Microsoft Image Composite Editor (version 1.4.4.0). Contrast was increased by using the “auto contrast” tool of Google Picasa (version 3.9.137) and Matlab’s “imadjust” function.

Mice were injected intraperitoneally with calcein (20 mg/kg body weight; C0875, Sigma–Aldrich) and alizarin-3-methyliminodiacetic acid (30 mg/kg body weight; A3882, Sigma–Aldrich) nine and two days, respectively, before euthanasia. Left femurs and vertebrae were fixed in 4% PFA in PBS for 48 h at room temperature, dehydrated with acetone, and embedded in methyl methacrylate without decalcification to obtain 7-µm sections. Goldner’s trichrome staining¹¹⁶ was performed for visualizing bone surface, osteoid, and osteoblasts, while tartrate-resistant acid phosphatase (TRAP) staining^{117 (link)} was used for osteoclast detection. Fluorescent labeling was assessed on unstained sections. Slides were imaged using ZEISS Axio Scan.Z1 scanner (Carl Zeiss). Static and dynamic histomorphometry was performed on the endosteal surfaces of the cortical femur diaphysis, and on the trabecular regions of the femur distal metaphysis and third lumbar (L3) vertebra under 20× magnification using BIOQUANT OSTEO analyzing software. Structural parameters (bone surface [BS], bone volume/tissue volume [BV/TV], trabecular diameter [Tb.Dm], trabecular number [Tb.N], and trabecular spacing [Tb.Sp]) were obtained by averaging measurements in three consecutive sections. All parameters were calculated and expressed according to standardized nomenclature.

Fluorescent dyes were used to follow dynamic calcium deposition over time (Fig. 2). Polyfluorochrome tracers, calcein green (30 mg/kg, C0875 SIGMA) and alizarin complexone (30 mg/kg, A3882 SIGMA) were injected intraperitoneally. They bind to calcium ions and later can be incorporated at mineralization sites in the form of hydroxyapatite crystals. The florescent label demarcates the mineralization front at the time of administration and can be detected in histological sections without any further staining or decalcification (Van Gaalen et al. 2010 (link); Rahn 2003 ). The rats were injected with calcein green 45 days after implantation and then with alizarin complexone 60 days after implantation. The animals were killed 63 days after implantation. The fluorescent dyes can be detected in histological sections with a fluorescent microscope (Nikon Eclipse Ti, Tokyo, Japan) with appropriate filters giving an indication of new matrix deposition over time (Kajiwara et al. 2005 (link)).Fig. 2

Time line showing the fluorochrome-labelling protocol