Immunohistochemical Analyses of Neuroinflammation

Immunohistochemical stainings were performed to assess (1) the amount of glucose transporter-1 (GLUT-1) as indicator for blood vessel quality and (2) activated microglia and macrophages as measure for neuroinflammation by ionized calcium-binding adapter molecule 1 (IBA-1). As described in detail previously, immunohistochemistry was performed using standard free-floating labeling procedures (Janssen et al., 2016 (link)). GLUT-1 was visualized using polyclonal rabbit anti-GLUT-1 antibody (1:40,000, Chemicon AB 1340, Chemicon International, Inc., Temecula, CA, United States) and as secondary antibody donkey anti-rabbit biotin (1:1500 Jackson ImmunoResearch, West Grove, PA, United States). For IBA-1 a primary antibody against IBA-1 polyclonal goat anti-IBA-1 (1:3000; Abcam, Cambridge, United Kingdom) was used. Donkey antigoat biotin (1:1500; Jackson ImmunoResearch) was used as a secondary antibody. Representative images of the ipsilateral and contralateral brain hemisphere were made with ImageJ (version 1.47, National Institute of Health, Bethesda, MD, United States) stitching plugin (Preibisch et al., 2009 (link)), using three images that were taken with 5× magnification of different ROI.