Horseradish peroxidase conjugated anti mouse antibody

Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokyo, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), in accordance with the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% DifcoTM skim milk (BD Biosciences, Franklin Lakes, NJ, USA) in 1× Tris-buffered saline (TBS, Bio-Rad, Hercules, CA, USA) with 0.1% Tween 20 (Sigma-Aldrich), and probed using various antibodies. The Western blots were visualized using ECL (Bio-Rad) and exposed to the Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: rabbit anti-BDNF (1:500, Abcam), rabbit anti-NGF (1:200, R&D Systems), mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), horseradish peroxidase-conjugated anti-rabbit antibody (1:2500, Abcam), and horseradish peroxidase-conjugated anti-mouse antibody (1:2500, Abcam).

Genomic DNA was extracted in each group and isolated using the DNeasy Blood & Tissue Kit (Qiagen) for the quantification of the 8-OHdG amount within genomic DNA. The purified genomic DNA samples were spotted on the nitrocellulose membrane (0.2 µm pore size). The DNA was immobilized to the membrane by baking at 80 °C for 2 h. The membrane was then blocked with 5% skim milk and incubated with mouse anti-dsDNA (1:2000, Abcam) and mouse anti-8-OHdG (1:200, Santa Cruz) at RT overnight. Horseradish peroxidase-conjugated anti-mouse antibody (1:1000, Abcam) was incubated for 1 h at RT and visualized by enhanced Clarity Max Western ECL Substrate (ECL, Bio-rad). The relative amounts of 8-OHdG in the genomic DNA were calculated using Amersham™ Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK).