Alizarin red

Live bone staining was performed using 0.2% (w/v) calcein or using 50 μg/ml alizarin red (Fisher Scientific; UK) as previously described (Kimmel et al., 2010 (link)). For cartilage and bone staining, we used alcian blue and alizarin red following the protocol outlined in (Walker and Kimmel, 2007 (link)) with modifications from protocols on www.zfin.org. Bone and cartilage staining in fixed larvae was performed on PFA fixed and then methanol dehydrated specimens, treated overnight at 4°C with 0.02% (weight to volume) alizarin red in 70% ethanol. Specimens were bleached (H₂O₂) and cleared before storing in glycerol for imaging.

Alizarin red staining of juvenile and adult zebrafish was performed as previously described [61 (link)]. Zebrafish were euthanized with 50 μg/mL benzocaine in 1 × E3 water and subsequently placed in bone-fixative solution (1.2 M formalin (Sigma-Aldrich, Australia), 0.1 M Triton X-100 (Promega, Fitchburg, WI, USA), 0.2 M potassium hydroxide (KOH)) for 24 h with rocking at 40 °C, then in enhancement solution (3.5 M ethylene glycol (Sigma-Aldrich, Australia), 0.1 M Triton X-100, 0.2 M KOH) for 24 h with rocking at 40 °C. Samples were then washed in distilled water for 5 min at room temperature (RT) before immersing into bone-staining medium (3.5 M ethylene glycol, 0.2 M KOH) for 15 min at RT. Next, samples were washed in bone-staining solution (0.2 M Alizarin red (Thermo-Fisher Scientific, Waltham, MA, USA), 3.5 M ethylene glycol, 0.2 M KOH) for 15 min with rocking at RT, followed by clearing solution (0.2 M Tween-20 (Thermo-Fisher Scientific, USA), 0.2 M KOH) for 24 h with rocking at 40 °C. Stained samples were stored in 4% PFA/PBS at 4 °C.

In addition, parallel experiments using six-well plates were also performed using DPSC isolates with and without the addition of laminin-5 and fibronectin. To evaluate any morphological changes and the deposition of calcium associated with osteogenic differentiation, each well was stained with Alizarin Red 1% w/v solution from ThermoFisher Scientific (Fair Lawn, NJ, USA) using the manufacturer’s recommended protocol. In brief, wells were washed with 1.0 mL of 1× PBS and then fixed. Media were aspirated and wells were stained with Alizarin Red for 15 min at room temperature. The stain was aspired and wells were washed three times with 1.0 mL of distilled water. Cells were visualized using an Axiovert inverted microscope from Zeiss (Hamburg, Germany).

Cells were seeded on 48-well culture plates (4×10⁴ cells/well) and were treated with FGF2 with or without U0126 every other day for 4 days. After removing FGF2-containing media, cells were cultured with osteogenic media (OM) (50 μg/mL of ascorbic acid, 5 mM of β-glycerophosphate, and 100 ng/mL of BMP2). The induction media was changed every 2 days. For alkaline phosphatase (ALP) enzyme staining, cells were fixed with 4% formaldehyde (Sigma-Aldrich) for 15 min and then treated with a BCIP/NBT solution (Sigma-Aldrich) for 30 min. To evaluate mineralization, cells were fixed with 70% cold ethanol and then treated with 40 mM of alizarin red (Sigma-Aldrich) solution (pH 4.2) for 30 min. The stained culture plates were scanned with an Epson Perfection V700 (Epson Korea, Seoul, South Korea), and the stained cells were observed via optical microscopy (magnification: 50×, Leica Microsystems, Wetzlar, Germany). For quantitative analysis, the alizarin red stain was extracted with 10% (w/v) cetylpyridinium chloride in 10 mM of sodium phosphate (pH 7.0) for 15 min and fluorescence was quantified by measuring absorbance at 540 nm using an ELISA reader (Thermo Fisher Scientific).