Immunohistochemical Analysis of Alzheimer's Pathology

The mice were anesthetized with avertin (0.25 g/kg via intraperitoneal injection) and perfused with PBS and neutrally buffered in 4% paraformaldehyde as previously described [14 (link)]. Brains were removed, postfixed over 6 h, and sectioned at a thickness of 40 μm. The sections were incubated with the following antibodies for 1–2 days at 4 °C: anti-Aβ_1–16 (6E10) (#803015, Biolegend, San Diego, CA, USA, 1:1000), anti-iNOS (sc-7271, Santa Cruz Biotechnology, Dallas, TX, USA, 1:20), and anti-ionized calcium-binding adapter molecule 1 (IBA-1) (ab5076, Abcam, Cambridge, UK, 1:200). The sections were washed and then incubated with Alexa Fluor 594- and/or 488-conjugated secondary antibodies (Invitrogen, 1:1000). The binding specificity of the primary antibodies was confirmed with secondary antibody-only controls. Digital images were obtained using a confocal microscope equipped with an Olympus Disk Spinning Unit (Olympus, Tokyo, Japan). Fluorescence intensity or signal-positive cell numbers were analyzed using ImageJ software (NIH, RRID:SCR_003070).