Ab96879

To visualize biotinylated (EIS)₂-RGD6 in animals used to perform experiment (EIS)₂-RGD6 II, a set of parallel sections per animal were first processed for the visualization of GFAP following the aforementioned fluorescence-based simple immunohistochemistry protocol. The following primary and secondary antibodies were used: monoclonal mouse anti-GFAP (G3893, Sigma-Aldrich, Steinheim, Germany; 1:500) and Dylight488-linked goat anti-mouse (ab96879, Abcam, Cambridge, UK). Subsequently, sections were immersed in buffer blocking—10% foetal bovine serum (10500064, Fisher Scientific, Madrid, Spain), 0.3% bovine serum albumin (A7906, Sigma Aldrich, Steinheim, Germany), 0.3% Triton X-100 (X100, Sigma Aldrich, Steinheim, Germany) in TBS—for 1 h at room temperature (RT), incubated with alexa594-linked streptavidin (S11227, Thermo Scientific, Paisley, UK; 1:500) for 1 h at RT and, after several washes, with DAPI (62247, Thermo Scientific, Paisley, UK; 1:5000) for 5 min at RT. Finally, sections were cover-slipped with Immumount (9990402, Epredia, Breda, The Netherlands).

NPCs were seed in 6-well plates. When cell number reached 5 × 10⁶/well, 200 μL RIPA reagent/well was used to extract the total proteins of NPCs in different treatment groups, and the proteins were quantitatively analyzed with BCA protein detection kit according to the instruction. The same amount of protein sample was separated through 12% SDS-PAGE, and then, the protein was electrically transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk powder, and then, the corresponding primary antibody was added for overnight incubation at 4 °C. The membranes were added with secondary antibody and incubated for 1.5 h. ECL solution was added for visualizing the bands, GAPDH was used as internal reference, and Image J software was used for quantitative analysis of protein gray. The primary antibodies used in the experiment included: MMP-3 (ab52915, Abcam, 1:1000), MMP-13 (ab51072, Abcam, 1:1000), ADAMTS-4 (ab84792, Abcam, 1:1000), ADAMTS-5 (ab41037, Abcam, 1:1000), collagen II (ab188570, Abcam, 1:1000), NOX4 (ab154244, Abcam, 1:1000), NF-κB (ab32536, Abcam, 1:1000), p-NF-κB (ab76302, Abcam, 1:1000), GAPDH (ab181602, Abcam, 1:1000). The second antibody used in the experiment included: Goat Anti-Rabbit IgG H&L (1:5000, ab96899, Abcam, Goat Anti-Mouse IgG H&L (1:5000, ab96879, Abcam).

Formalin-fixed samples were decalcified in Calci-Clear Rapid solution (National Diagnostics, GA, USA) for 24 h at RT. Specimens were dehydrated through increasing concentrations of ethanol, followed by paraffin embedding. Samples were sectioned at a thickness of 7-μm and stained with hematoxylin-eosin (H&E), safranin O, and toluidine blue according to standard protocols. Immunohistochemistry for GFP, type II collagen, and type I collagen in BMSCs-laden samples was performed using primary antibodies for GFP (sc-9996, dilution ratio 1:200, Santa Cruz Biotechnology), anti-collagen type II (ab34712, dilution ratio 1:200, Abcam), and type I collagen (NBP2-46874, dilution ratio 1:100, Novus Biologicals, CO, USA). Secondary antibodies were Goat Anti-Mouse IgG H&L DyLight 488 (ab96879, dilution ratio 1:500, Abcam) and Goat anti-Rabbit lgG (H + L) DyLight 594 (35561, dilution ratio 1:1000, Invitrogen). Quantitative histomorphological analysis was carried out by five blinded researchers to evaluate osteochondral regeneration using the modified O’Driscoll scoring system⁵⁷ .

The hESCs from human skin were purchased from Otwo Biotech Inc. (Shenzhen, China) and cultured in EpiLife medium (Gibco, M-EPI-500-CA) supplemented with 1% human keratinocyte growth supplement (HKGS) (S-001-5). Cell phenotype was detected by flow cytometry. Briefly, after the cells were fixed, permeabilized and blocked, they were incubated with CK19 and ITGβ1 for 30 min, followed by incubation with goat anti-mouse secondary antibody for 30 min. The fluorescence was detected by a flow cytometer. The antibodies including anti-cytokeratin 19 (CK19; ab7754), anti-integrin beta-1 (ITGβ1; ab24693) and goat anti-mouse IgG (H + L) (ab96879) were obtained from Abcam.