Zli 9010

For histological analysis, harvested mouse joints were fixed with 4% PFA for 2 days and embedded in paraffin after decalcification for 14–21 days. Sections (5 μm) were stained with fast green FCF (0.02%) and safranin O (0.1%) according to the manufacturer’s instructions.
Immunohistochemical staining was performed using the DAB staining method as previously described, with some modifications.^{33 (link),34 (link),102 (link)} First, slides were deparaffinized and rehydrated using xylene and different concentrations of alcohol. Antigen retrieval was performed using Trypsin (ZLI-9010, ZSGB-BIO) digestion for 20 min at room temperature. Hydrogen peroxide (3%) was used to block endogenous peroxidase activity by incubating for 10 min at room temperature. Slides were then blocked with 10% donkey serum for 1 h at room temperature and incubated with an anti-P16 antibody (Ab54210, Abcam, 1:200) at 4 °C overnight and with a secondary antibody (PV-6002, ZSGB-BIO) for 1 h at room temperature before DAB staining (ZLI-9017, ZSGB-BIO).

Before staining, slides were baked at 65°C for 2 h. After deparaffinized and rehydrated, slides were subjected to antigen repair with 0.2% parenzyme (ZLI-9010, ZSGB-BIO, China) at 37°C for 15 min. Then the slides were stained according to the manufacturer’s instructions (SP-0022, Bioss, China). After PBS washed, slides were incubated with 0.3% hydrogen peroxide at 37°C for 20 min. After PBS washing, slides were incubated with goat serum at 37°C for 20 min to block reagent. Drain the slides and carefully wipe off the excess serum blocking reagent (do not rinse with PBS).
Slides were then incubated with anti-MPO primary antibody (Bioss, China, 1:100) at 4°C overnight. After PBS washed, the slides were incubated with goat anti-rabbit secondary antibody for 20 min at 37°C. After PBS washed, incubate samples with 1-3 drops of HSS-HRP for 20 min at 37°C. After PBS washed, the slides were then stained with 3,3-diaminobiphenyl tetrasodium (DAB) substrate for 20 s, and counterstained with hematoxylin. The staining images were scanned and obtained with Olympus scanner (VS-200, Japan). Count the MPO + cells (neutrophils, macrophages) in two random 100 × 100 μm² square areas of the gingiva and periodontal ligament between the second molar and the first molar. The investigator was blinded to the groups while the data measurement and outcome evaluation were being performed.