8 hydroxy 2 deoxyguanosine 8 ohdg

Terminally anesthetized mice were perfused intracardially with saline followed by 4% paraformaldehyde. The fixed brains were embedded in paraffin and cut into serial 6 μm thick coronal slides. Immunohistochemistry was performed with antibodies against Iba-1 (Wako Chemicals USA Inc., Richmond, VA, USA) to identify macrophages and microglia; 4-hydroxy-2-nonenal (4-HNE; Abcam, Cambridge, MA, USA) and 8-hydroxy-2-deoxyguanosine (8-OHdG; Abcam) to identify lipid peroxidation and damaged DNA of oxidative impairment. Immunolabeling was detected by applying the peroxidase-antiperoxidase procedure with 3,3′-diaminobenzidine as a co-substrate. For double immunofluorescent staining, antibodies against NeuN (Millipore, Billerica, MA, USA) and cleaved Caspase-3 (Cell Signaling, Danvers, MA, USA) were used to identify apoptotic neurons. Respective negative controls that omit primary antibodies and positive controls were applied for each case. The positive cells were counted at 20× magnification in matched sections. Results are presented as Iba-1⁺, 4-HNE⁺, 8-OHdG⁺ or cleaved-Caspase-3⁺/NeuN⁺ cells per mm² within areas measured from 20× images using Image J.1.34vi software (National Institutes of Health) [12 (link)].

Paraffin sections (4 μm) of TA muscle tissues were rehydrated and microwaved in citric acid buffer to retrieve antigens. After incubation with 10% BSA for 1 h, the sections were respectively incubated with primary antibodies against CD45 (1:200; Abcam, MA, United States) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) (1:200; Abcam) overnight. Subsequently, tissue slices were incubated with secondary antibody corresponding to each primary antibodies in dark for 1 h and counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Jiangsu, China). Stained slices were observed using a laser scanning confocal microscopy.

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.