Histochemical Analysis of Oxidative Stress and Inflammatory Markers in Periodontal Tissues

Periodontal sections were blocked in 10% normal goat serum; incubated overnight with primary antibodies against 3-nitrotyrosine (3-NT) (1:200) (Abcam, Cambridge, UK), 8-Hydroxy-2'-deoxyguanosine 8-OHdG (1:200) (Abcam), Nrf2 (1:300) (Abcam), and HO-1 (1:300) (Abcam), Interleukin (IL)-6 (1:200) (Bioss), tumor necrosis factor alpha TNF-α (1:100) (Bioss), receptor activator of NF-ҡB ligand RANKL (1:300) (Abcam), alkalinephosphatase ALP (1:300) (Abcam) and osteocalcin OCN (1:200) (Abcam) at 4 °C; and stained with HRP-conjugated secondary antibodies (ZSJQ-BIO, Beijing, China). Diaminobenzidine (DAB) was used as a substrate for color development. All slides were counterstained with hematoxylin. Images of histologically stained sections were obtained using a BX41 microscope (Olympus, Japan). The H-score method 29 (link) was applied for calculating the staining score of each sample, which was to multiply the immunoreaction intensity (negative: 0, weak: 1, moderate: 2, strong: 3) by the staining extent score (0%-100%). According to the H-score, the stained samples were divided into four groups: negative (-; 0), weak (+; 0~1), moderate (++; 1~1.5), and strong (+++; 1.5~3). Samples with a negative or weak H-score were determined to be the low protein expression group, whereas those with a moderate or strong H-score were classified as the high protein expression group.