Mab1419

NFs were harvested and fixed for 2 h in 10% buffered formalin at room temperature and prepared for paraffin embedding. To detect calcium deposition, sections were stained with 5% silver nitrate solution (ID Labs Biotechnology, Buffalo, NY), exposed to ultraviolet light for 30 min, and stained with nuclear fast red solution (ID Labs Biotechnology). To detect matrix proteoglycans, sections were stained with 0.1% Safranin O solution (Muto Pure Chemicals, Tokyo, Japan) and counter-stained with hematoxylin.
The primary antibodies used for immunohistochemistry included monoclonal mouse anti-human runt-related transcription factor 2 (RUNX2; H00000860-M06, Abnova, Taipei, Taiwan, 1: 100 dilution), monoclonal mouse anti-human osteocalcin (MAB1419, R&D, Minneapolis, MN, 1: 1000 dilution), monoclonal mouse anti-human dentin matrix protein-1 (DMP-1; sc-73633, Santa Cruz, Dallas, TX, 1: 100 dilution), and polyclonal rabbit anti-human type-II collagen antibody (ab34712, Abcam, Cambridge, MA, 1: 100 dilution). The secondary antibodies were horseradish peroxidase (HRP)-conjugated antibodies (Simple stain MAX PO, Nichirei, Tokyo, Japan). Antigens were visualized using a 3,3-diaminobenzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark) and counter-stained with hematoxylin.

Prior to staining, the microtome sections were deplasted in 100% xylene for one minute. Next, the cells were permeabilized with 0.1% Triton X-100 for 10 min. To prevent nonspecific antibody binding, the sections were then blocked with 2% bovine serum albumin (BSA) for 1 h and washed with PBS for 5 min. The primary antibody (anti-OCN, monoclonal mouse antibody, isotype: IgG1, MAB1419; R&D Systems, Minneapolis, MN, USA) was diluted to a concentration of 10 μg/mL in 2% BSA and stored overnight at 4 °C in a humid chamber. The next day, the sections were washed with PBS, and a DyLight405-labeled secondary antibody (polyclonal goat anti-mouse IgG1 antibody, 409109; Biolegend, San Diego, CA, USA) was diluted to a concentration of 5 μg/mL in 2% BSA and incubated for one hour. The sections were then washed with PBS, and a nuclear staining solution with Sytox Orange (5 mM stock solution diluted 1:1000 in PBS, ThermoFisher Scientific, Waltham, MA, USA) was incubated on the slides for 10 min. Fluorescence intensity of ALP and OCN immunostaining was quantified using ImageJ software and normalized to the intensity of the nuclear staining.