Vectastain pk 400

Constructs were fixed in 4% paraformaldehyde, dehydrated in a graded ethanol series, embedded in paraffin wax, sectioned at 8 µm and affixed to microscope slides. The sections were stained with aldehyde fuschin/alcian blue to assess sGAG content and Picrosirius red to assess collagen content. Collagen types II and X were evaluated using a standard immunohistochemical technique; briefly, sections were treated with peroxidase, followed by treatment with chondroitinase ABC (Sigma-Aldrich, Dublin, Ireland) in a humidified environment at 37°C to enhance permeability of the ECM. Sections were incubated with goat serum to block non-specific sites and collagen type II (ab3092, 1:100; 1 mg/mL) or collagen type X (ab49945, 1:200; 1.4 mg/mL) primary antibodies (mouse monoclonal, Abcam, Cambridge, UK) were applied for 1 h at room temperature. Next, the secondary antibody (anti-mouse (Immunoglobulin G) IgG biotin conjugate, 1:200; 2.1 mg/mL) (Sigma-Aldrich, Dublin, Ireland) was added for 1 h followed by incubation with ABC reagent (Vectastain PK-400; Vector Labs, Peterborough, UK) for 45 min. Finally, sections were developed with (3, 3’-diaminobenzidine) DAB peroxidase (Vector Labs) for 5 min. Positive and negative controls were included in the immunohistochemistry staining protocol for each batch.

The hMSCs and PKH26-hMSCs were seeded onto 6-well plates, and differentiation of these cells into chondrocytes and adipocytes was induced at 40–50% confluence. To induce chondrocyte differentiation, cells were cultured with chondrogenic differentiation medium comprising 1.5×10⁻⁴ mg/ml ascorbic acid and 1 ng/ml human recombinant transforming growth factor-β (Sigma-Aldrich). Immunohistochemistry was performed to examine the presence of types II collagen. Cell-seeded constructs were rinsed with 1X phosphate-buffered saline, fixed in 4% formalin for 24 h and embedded in paraffin. Slides were incubated with 10% normal goat serum to block non-specific sites and mouse monoclonal collagen type II (1:100; 1 mg/ml; cat. no. ab3092; Abcam, Cambridge, UK) primary antibodies were applied for 1 h at 22°C. Secondary antibody (anti-mouse immunoglobulin G biotin conjugate; 1:200; 2.1 mg/ml; cat. no. B7151; Sigma-Aldrich) was added for 1 h followed by incubation with ABC reagent (Vectastain PK-400; Vector Laboratories, Inc., Peterborough, UK) for 45 min. To induce adipocyte differentiation, cells were cultured with adipogenic differentiation medium comprising 1×10⁻⁸ mol/l dexamethasone and 1×10⁻¹⁰ mol/l insulin (Sigma-Aldrich) (8 (link)). Two weeks after differentiation, adipocytes were identified by the existence of lipid vesicles, using staining with Oil Red O (Sigma-Aldrich).