Immunofluorescence Staining of Periodontal Membrane

For immunofluorescence staining, paraffin-embedded sections were immersed in 0.3% H₂O₂ for 30 min and blocked in 1% BSA-PBS for 20 min. Double immunofluorescence staining was performed on sections to localize Treg cells, Th17 cells, p-STAT3⁺IL-17A⁺ cells and p-STAT5⁺FOXP3⁺ cells. Sections were incubated overnight at 4°C with the following mixed antibodies: (Tonetti et al., 2018 (link)) anti-IL-17A antibody (1:100; ab189377, Abcam) and anti-CD4 antibody (1:300; ab183685, Abcam), (Cecoro et al., 2020 (link)) anti-FOXP3 antibody (1:100; ab253297, Abcam) and anti-CD4 antibody (1:300; ab183685, Abcam), (Barutta et al., 2022 (link)) anti-IL-17A antibody (1:100; ab189377, Abcam) and anti-p-STAT3 (1:100; ab76315, Abcam), (Zheng et al., 2021 (link)) anti-FOXP3 antibody (1:100; ab253297, Abcam) and anti-p-STAT5 antibodies (1:100, AF3304, Affinity Biosciences, United States). Sections were incubated with mixed green fluorescent goat anti-rabbit IgG H&L (Alexa Fluor 647) (1:200; ab150081, Abcam) and red fluorescent goat anti-mouse IgG H&L (Alexa Fluor 488) (1:200; ab150119, Abcam) antibodies at room temperature in the dark for 1 h. After washing three times in PBS, samples were nuclear stained with 4′,6-diamidino-2-phenylindole (ab104139, Abcam) for 5 min. Images were obtained under inverted fluorescence microscopy (DMi8 S; Leica, Germany) and the periodontal membrane near the root of the maxillary second molar was selected as the area of interest. All cells were counted at × 400 magnification of the original magnification in immunofluorescence images.