Nonspecific staining blocking reagent

To elucidate the expression of ACAN (1:200; ab36861, Abcam, Cambridge, United Kingdom), MMP‐9 (1:1000; ab38898, Abcam), MMP‐13 (1:1000; ab39012, Abcam), interleukin (IL)‐1β (1 /ml; ab9722, Abcam), and cleaved caspase‐3 (1:100; 9509S, Cell Signaling Technology, Danvers, MA), and apoptosis marker in the mandibular condylar cartilage, immunohistochemical staining was carried out using several primary antibodies (Immuno Biological Laboratories, Fujioka, Japan). After deparaffinization and blocking, the sections were incubated overnight with primary antibodies diluted in phosphate‐buffered saline/0.1% bovine serum albumin 4°C in a humid atmosphere. Immunostaining was performed using a Histofine simple stain kit (Nichirei, Tokyo, Japan). Briefly, after blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol, non‐specific binding of the antibody was blocked by incubating the section for 30 min with non‐specific staining blocking reagent (Dako, Carpinteria, CA). After washing with PBS, the sections were incubated with the corresponding secondary antibodies for 1 hr at room temperature. Finally, immunoactivity was detected using diaminobenzidine, followed by counter‐staining with Mayer's hematoxylin. The sections were observed under a BioRevoBZ‐9000 microscope (KEYENCE, Osaka, Japan).

Sections with a thickness of 4 μm were obtained from the paraffin-embedded blocks. After incubation at 60 °C for 10 min, the sections were deparaffinized using xylene and rehydrated using a graded series of ethanol. The slides were subsequently washed twice for 5 min in phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked for 15 min using absolute methanol containing 3% hydrogen peroxide. After washing the sections in PBS, they were microwaved for 10 min to achieve antigen retrieval. Non-specific binding was blocked using a non-specific staining blocking reagent (Dako, Kyoto, Japan). The sections were then incubated overnight at 4 °C with mouse monoclonal antibodies (CD15 or C-X-C motif chemokine receptor 2 (CXCR2), 1:100 dilution; Abcam, Tokyo, Japan), and subsequently washed using PBS for 10 min before a 10-min incubation with a secondary antibody at room temperature. After washing the sections using PBS, they were visualized using 3-3′-diaminobenzidine for 5 min and then counter-stained using haematoxylin before mounting.