Alcian blue

Euthanized wild-type and Dyrk2^-/- mice at E18.5 and E16.5 were skinned, eviscerated, and fixed in 100% EtOH. For skeletal analysis, the embryos were stained with 1% Alcian Blue (Wako Pure Chemicals, Osaka, Japan) dissolved in 20% glacial acetic acid and 80% EtOH and 0.01% Alizarin Red (Sigma-Aldrich, St. Louis, MO) dissolved in 1% KOH. The excised tissues were observed using a stereo microscope (BioTools, Gunma, Japan). Ten embryos of each wild-type and Dyrk2^-/- mice were analyzed.

Colonies were dissociated using Accutase (Nacalai Tesque) and reseeded on 35-mm tissue-culture dishes. Cells were cultured to 70–80% confluence with MesenCult Proliferation medium. To induce differentiation into adipocytes and osteoblasts, cells were cultured using the MesenCult Adipogenic Differentiation Kit (Mouse) or MesenCult Osteogenic Stimulatory Kit (Mouse) (Veritas) for 3 weeks. Cells were then fixed with 4% PFA/PBS. Alizarin Red (40 mM; Sigma) and Oil Red O (0.5%; Wako) staining was performed. To induce differentiation into chondrocytes, cells were cultured in round-bottom 96-well plates at 1 × 10⁴ cells/well, and cell aggregates were allowed to form for 2 days. After 2 days, these aggregates were cultured with Chondrogenic Differentiation Medium (R&D Systems, Inc., Minneapolis, MN, USA) for 3 weeks. Aggregates were then fixed with 30% ethanol and 2% sucrose/PBS at 4°C overnight and then incubated with 60% ethanol, 40% acetic acid, and 0.1% Alcian Blue (Wako) for 2 h. After incubation, these aggregates were washed with 55% ethanol and 35% acetic acid solution three times.

hDPSCs (1 × 10³ cells) seeded in spheroid-forming culture plates (PrimeSurface; Sumitomo Bakelite, Tokyo, Japan) were cultured in a chondrogenic differentiation medium (Mesenchymal Stem Cell Chondrogenic Differentiation Medium; PromoCell, Heidelberg, Germany) for 25 days. Alcian Blue (Fujifilm Wako Pure Chemical) staining was performed on the frozen sections (5 μm thickness).