Abundant Iba1/AIF-1 positive parenchymal and perivascular macrophage/microglia (red color) costain for osteopontin (OPN) (brown color) expression in tissue from the occipital lobe of an HIV-infected individual with asymptomatic neurocognitive impairment (ANI). Paraffinembedded human autopsy tissue from the occipital lobe (National NeuroAIDS Tissue Consortium). Antigen retrieval was performed in citric acid buffer pH 6.0 and slides were stained sequentially with rabbit polyclonal antisera against Iba1/AIF-1 (SIGMA) overnight at 4°C followed by incubation with goat-anti-rabbitalkaline phosphatase (AP) secondary for 1 hr at room temperature and developed with permanent FastRed Quanto (ThermoFisher) (red color). Slides were then incubated with mouse monoclonal antibody to OPN (MAB194, Maine Biotechnology) at room temperature for 2 hrs followed by goat anti-mouse-horse radish peroxidase for 1 hr and developed with 3,3’-diaminobenzidine (brown color). Images were taken on an Axio Observer A1 inverted microscope (Zeiss) at 20x magnification. Adjustment of the image brightness, contrast and sharpness was performed with Adobe Photoshop 5.5 using the same settings for each image.