Alizarin red s

To identify the wnt4b-mutant among the offspring produced by progeny tests using wild type recipients that received the above-mentioned mutant germ cells, skeleton staining was performed. In particular, bony tissues were stained with Alizarin Red, and cartilage was stained with Alcian blue38 (link)39 (link). Specimens were fixed for 3 days in fixing solution (10% formalin in phosphate-buffered saline) at room temperature. The fixed specimens were washed 3 times in PBS and then their cartilage was stained in 0.1% Alcian blue solution diluted in 70% (v/v) ethanol and 30% (v/v) glacial acetic acid (Sigma) for 30 min. After stepwise washing with 95%, 75%, 50%, and 25% ethanol and distilled water including 30% sodium tetraborate decahydrate, the specimens were immersed in 30% sodium tetraborate decahydrate solution containing 10 mg mL⁻¹ trypsin (Worthington, Lakewood, US) at 37 °C overnight to make them transparent. The specimens were washed three times in 0.5% KOH and their skeleton was stained with the solution containing 4% Alizarin Red S (Wako) at 23 °C for 2 h. The stained samples were kept in glycerol and their vertebral column was observed under a binocular microscope (BX-51-34FL, Olympus).

Fish were fixed with 10% formalin for 7–10 days and their cartilages were stained for 30 min with 1 mg/ml alcian blue 8GX solution (Sigma-Aldrich) diluted in 70% ethanol and 30% glacial acetic acid. The specimens were then hydrated in a degraded ethanol series (95, 75, 50, and 25%) and immersed in 30% sodium borate solution containing 0.1 g/ml trypsin (ThermoFisher Scientific) for 16–24 h at 37 °C. After washing with 0.5% aqueous KOH, the endoskeletons were stained for 12 h with 20% Alizarin Red S (FUJIFILM Wako Pure Chemical Corporation) solution containing 0.4% KOH. The specimens were then immersed in a series of glycerin-0.5% KOH solutions (25, 50, and 75% glycerin) for several days each and preserved in pure glycerin containing 0.025% sodium azide. The numbers of vertebrae and fin rays were determined under a stereomicroscope (SZX-10, Olympus).

Cells were plated in 96-well plates at a density of 1×10⁴ cells/well, and cultured for 4 days in the medium described above in the presence or absence of BMP-2 (300 ng/mL), followed by additional 3-day culture in DMEM supplemented with 2.5% FBS, ascorbic acid (50 μg/mL), β-glycerophosphate (10 mmol/L), and dexamethasone (10 nmol/L). Cells were washed with PBS, fixed in 95% methanol, stained with 1% alizarin red S (pH 6.3–6.5) (Wako Pure Chemicals) for 5 minutes, and washed with water to remove unbound dye. Calcified nodules visualized by alizarin red S were observed under a microscope. For evaluation of calcium deposition, alizarin red S bound to cell matrices was dissolved in 10% (w/v) cetylpyridinium chloride in water and quantified by measuring absorbance at 570 nm.

At day 21 of cell culture, HC300 was collected from the medium and washed with PBS five times. Calcified depositions formed on HC300 were stained with Alizarin Red S (FUJIFILM Wako Pure Chemical Corporation). Pure HC300 before cell seeding was stained with Alizarin Red S as a control.

The hMSCs were cultured with MSC-Exo (5 μg/mL) + DMEM(-) (MSC-Exo), MSC-Exo (5 μg/mL) + anti-VEGFA antibody (100 ng/mL) + DMEM(-) (Exo-antiVEGF), MSC-CM, or DMEM with 5% FBS for 14 days. Each medium was replaced every 3 days. After 14 days, hMSCs were washed twice in PBS and fixed in 10% neutral formalin for 15 minutes. The cells were stained with alizarin red S (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) for 30 minutes. The area of the mineralized matrix depositions was observed by light microscopy (ECLIPSE TS100, Nikon Co., Tokyo, Japan).

Polychrome sequential labeling of bone via intraperitoneal injection of fluorescent dyes was employed to determine the process and characteristics of new bone formation and mineralization after implantation according to the following timetable: rats were injected with 25 mg/kg oxytetracycline hydrochloride (Sigma-Aldrich, USA) at 1 week after implantation, with 30 mg/kg alizarin red S (011-01192, Wako, JP) at 4 weeks, and with 20 mg/kg calcein (340-00433, Wako, Japan) at 8 weeks. Rats were then anesthetized and euthanized at 8 weeks, and the right femurs including the implants were placed in a saline solution immediately after dissection and scanned with an SMX-130CT microcomputed tomography (micro-CT) scanner (Shimadzu) operated at 90kV and 40 μA with a copper filter. Three-dimensional reconstruction models were obtained using morphometric software (TRI/3D-BON; Ratoc System Engineering, Tokyo, Japan). The region of interest was defined as 2 mm below the highest point of the growth plate and extending 500 μm around each implant. The bone volume fraction (BV/TV), mean trabecular number (Tb.N), mean trabecular thickness (Tb.Th), and mean trabecular separation (Tb.Sp) were quantified to assess bone regeneration.

Zebrafish embryos were dechorionated and mounted in 1% low-melting agarose on a 35 mm glass bottomed dish (Asahi Techno Glass) with 0.016% tricaine (Sigma-Aldrich) in fish medium as described previously48 (link). The dish was submerged in fish medium containing 0.001% tricaine. Bone staining with alizarin red s (Wako) was performed as per the manufacturer’s protocol.
Confocal images were obtained with an FV1000 confocal upright microscope system (Olympus) equipped with a 20× water-immersion lens (XLUMPlanFL, NA 1.0). 473 nm and 559 nm laser lines were employed for green fluorescence protein (GFP and Kaede-green), and red fluorescence molecules (mCherry and alizarin red s), respectively. Image files were processed and analyzed using FLUOVIEW Viewer software (Olympus), MetaMorph (Molecular Devices), and Volocity (perkinelmer).