Anti 8 oxo dg monoclonal antibody

After fixing the specimens in 4% paraformaldehyde, they were embedded in a paraffin bath. After blocking endogenous proteins, sections were incubated overnight at 4°C with anti-8-oxo-dG monoclonal antibody (1 : 1000; Trevigen, Gaithersburg, USA). After washing the sections with PBS, they were incubated with Cy3-labeled secondary antibodies (1 : 2000; Beyotime Biotechnology, Shanghai, China) at 37°C for 1 h. Then, the slides were observed and pictures were captured under a fluorescence microscope.

HaCaT cells were cultured on coverslips and fixed in 4% paraformaldehyde for 15 min. Then, they were permeabilized with 0.5% Triton X-100 for 10 min. This blocked the nonspecific binding with PBS, which contained 1% bovine serum albumin (BSA) for 30 min. Then, they were incubated overnight with anti-8-oxo-dG monoclonal antibody at 4°C (1 : 800; Trevigen, Gaithersburg, USA). Subsequently, cells were washed again and incubated with Cy3-conjugated secondary antibody (1 : 1000, Beyotime Biotechnology, Shanghai, China) for 2 h. After washing stained cells with PBS, photomicrographs were taken with a fluorescence microscope (Nikon, Tokyo, Japan). The 8-oxo-dG-positive nuclei appeared red in color.

The biomarker 8-oxo-dG was detected in the mammary glands and liver tissues through ELISA and IHC analyses. For ELISA, 50 mg of the tissues was collected. Total DNA was extracted using a genome extraction kit (Sigma-Aldrich) and quantified by colorimetric method. Approximately 300 ng of DNA was extracted from each sample. The content of 8-oxo-dG in the total DNA was determined using the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) (EpiGentek). The results are presented as the ratio of 8-oxo-dG-positive cells.
For IHC, mammary glands were fixed in 10% formaldehyde for at least 24 h and sliced into 4 μm sections after embedding in wax. After dewaxing, hydration, antigen retrieval, DNA hybridization, and sealing treatment, the slices were incubated overnight in anti-8-oxo-dG monoclonal antibody (1 : 250, Trevigen) at 4°C. Next, the slices were incubated in DyLight 594-labled goat anti-mouse IgG (1 : 250, Jackson) for 30 min. After washing, the slices were evaluated using a fluorescence microscope.