8 ohdg

IHC analysis was performed as described previously^{8 (link)}. Slides were incubated with primary antibodies against Ki-67 (Thermo Fisher Scientific, MA, USA), 8-OH-dG (Abcam, Cambridge, UK), 4-HNE (Abcam), Nrf-2 (Abcam), NFκB p65 (Cell Signalling Technology, MA, USA), IL-1β (Abcam), TNF-α (Abcam) or MPO (Abcam) overnight at 4 °C. Thereafter, tissues were incubated with biotin-linked secondary antibody followed by signal detection using streptavidin horseradish peroxidase (HRP) conjugate (Thermo Fisher Scientific). Quantitative analysis of the immunolabelled tissues was done in a blinded fashion by counting the number of positively stained cells out of the total number of cells per field at 400 × magnification using Image J software version 1.46r (NIH, Bethesda, Maryland, USA).

Mice were sacrificed, and their brains were post-fixed in 4% paraformaldehyde, dehydrated with 30% sucrose at 4 °C overnight and then embedded in paraffin. Sections were cut to a thickness of 10 μm on glass slides. The sections were blocked with 10% donkey serum for 1 h, incubated with mouse anti-IBA-1 (Proteintech, USA) or 8-OH-dG (Abcam) antibodies overnight at 4 °C, and incubated with secondary antibodies for 1 h at room temperature. After being washed, the sections were incubated with 4’,6-diamidino-2-phenylindole for nuclear staining.
Three fields and five sections were used in this experiment, and the CA1 region of the hippocampus was analyzed. The area of selected cells was converted into a binary image. Total immunoreactivity was calculated as the percentage area density, defined as the positively stained area divided by the sum of the positively and negatively stained areas in the image field. In the IBA-1 analysis, microglial activation was expressed as the cell body/cell size of IBA-1-stained microglia [52 (link)]. The pictures are shown at 400× magnification. Images were captured with a microscope and analyzed with ImageJ software (National Institutes of Health).

After deparaffinization, sections were incubated with primary antibodies against polyclonal anti-LC3B (#2775, Cell Signaling Technology. Danvers, MA, USA), LAMP-1 (sc-17768, Santa Cruz Biotechnology, Santa Cruz, CA, USA), collagen-III (sc-271249, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 8-OHdG (ab62623, Abcam, Cambridge, MA, USA), and MDA (MDA11-S, Alpha Diagnostic International. San Antonio, TX, USA). Then, biotin-conjugated secondary IgG (1:200 dillution, Vector Laboratories, Burlingame, CA, USA), an avidin–biotin–peroxidase complex (ABC Elite Kit; Vector Laboratories), and DAB were added. Finally, the sections were visualized under a light microscope, and the digital images were analyzed (Elements BR3.2, Nikon, Japan).

After deparaffinization, the sections were incubated with primary antibodies against monoclonal anti-ICAM-1 (BD Bioscience, Franklin Lakes, NJ, USA), MCP-1 and polyclonal anti-F4/80 (Santa Cruz), MCP-1 (Santa Cruz), 8-OHdG (Abcam, Cambridge, UK), followed by biotin-conjugated secondary IgG (diluted 1:200; Vector Laboratories, Burlingame, CA, USA), avidin–biotin–peroxidase complex (ABC Elite Kit; Vector Laboratories), and DAB. Next, we visualized the sections by light microscopy and captured and analyzed digital images using NIS-Elements BR 3.2 (Nikon, Japan).