Red fluorescent conjugated secondary antibody

For morphological analyses, brainstems were removed and postfixed in 4% paraformaldehyde for 72 h at 4 °C after saline perfusion. Next, the samples were cryoprotected in 30% sucrose solution and stored at − 20 °C for further immunohistochemical staining. For immunostaining, brainstems were sliced with a freezing microtome at 30 μm. Samples were collected in cryoprotectant (30% ethylene glycol, 20% glycerol, 50 mmol/L sodium phosphate buffer, pH 7.4). After washing with PBS buffer and permeabilization with 0.1% Triton X 100 in 0.1% sodium citrate, the 4% paraformaldehyde fixed brain slices were incubated with rabbit anti-8-hydroxyguanosine (1: 200, Abcam) in 10% goat serum PBS buffer. The slices were then incubated with green fluorescent-conjugated secondary antibody (Thermo Fisher Scientific Inc.) for 1 h. After washing, the slices were incubated with rabbit anti-NeuN (1: 500, Millipore) in 10% goat serum PBS buffer followed by a red fluorescent-conjugated secondary antibody (Thermo Fisher Scientific Inc.) for another 1 h. DAPI was used as the blue nuclear stain. The expression and distribution of fluorescent signals were detected under an FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).

Brain sections from all groups were prepared as described in Section 2.3.2; then, endogenous peroxidase activity was blocked with 0.3% H₂O₂ for 10 min, washed in PBS, blocked with 5% BSA for 1 h, then species were incubated with the primary antibody (mouse 8-OHdG 1 : 200 in 10% goat serum PBS buffer) overnight at 4°C. The slices were then incubated with red fluorescent-conjugated secondary antibody (Thermo Fisher Scientific Inc.) for 1 h. After washing, the slices were incubated with rabbit anti-NeuN (1 : 1000 in 10% goat serum PBS buffer) followed by a green fluorescent-conjugated secondary antibody (Thermo Fisher Scientific Inc.) for another 1 h. Sections were then washed in PBS, and DAPI was used as the blue nuclear stain. Sections were viewed and processed in a Leica scanning confocal microscope. NeuN-positive cells were counted using ImageJ software (NIH) with a DAPI counterstain. The relative intensity of fluorescence in NeuN-positive cells/field of view was used for statistical analysis.