Anti runx2

Mid-sagittal sections of lumbar spines were used for immunohistochemical analyses. Antigen retrieval was performed in 0.1% (v/v) Triton-X followed by incubation in 3% (v/v) hydrogen peroxide in methanol. Slides were blocked in species-specific serum (5%) in PBS for 30 min at room temperature, followed by primary antibody incubation in a humidified chamber overnight at 4 °C. Primary antibodies were diluted in blocking solution as follows: rabbit polyclonal anti-COLX (1:750; abcam, Cambridge, England; ab58632); rabbit polyclonal anti-MMP13 (1:200; Proteintech, Rosemont, IL, USA; 18165-1-AP); rabbit polyclonal anti-RUNX2 (1:100; Novus Biologicals, Centennial, CO, USA; NBP1-77461). As negative control, slides were incubated overnight in blocking solution in the absence of primary antibody and IgG isotype controls were included for MMP13 and RUNX2 immunostaining. Slides were incubated in secondary antibody diluted in PBS [goat anti-rabbit for RUNX2 immunodetection (1:100; R&D Systems, Minneapolis, MN, USA; HAF008), goat anti-rabbit for MMP13 and COLX immunodetection (1:250; SantaCruz, Dallas, TX, USA; sc-2004)] for 1 h at room temperature. Secondary antibodies were conjugated with horseradish peroxidase and visualized following incubation with diaminobenzidine substrate (Dako Omnis, Santa Clara, CA, USA), followed by counterstaining with 0.5% methyl green.

The protein expression of odontogenic-related genes, including dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (Runx2), was detected by Western blot analysis. The DPSCs were appropriately cultured in an odontogenic medium containing either 20 µg/mL CM-EV or OM-EV for 7 and 14 days as explained above. Cells were then collected and lysed in RIPA buffer (KeyGen BioTECH, Nanjing, China) containing a 1% protease inhibitor cocktail (CWBIO, China). The protein concentration was determined by employing the BCA assay kit (CWBIO). The proteins of each group were separated by 10% SDS-PAGE (GenSpirt, China) and transferred to PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk, the PVDF membranes were subsequently incubated overnight at 4 °C with primary antibodies anti-DSPP (1:500, Novus, USA), anti-DMP-1 (1: 500, Bioss), anti-Runx2 (1:1000, Novus), anti-ALP (1:1000, Novus), anti-GAPDH (1:1500, Novus), followed by incubation with secondary antibody (1:5000, GNI, Japan). An advanced chemiluminescence detection system (Millipore) was utilized to detect immunoblots with an imaging system (ChemiDoc, Bio-Rad, China). The bands’ intensities were suitably measured via ImageJ and normalized to GAPDH.