Donkey antigoat biotin

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.

From paraformaldehyde-fixed tissue, 7 μm sections were prepared, as previously described (Gray et al., 2012 (link)). In some cases, sections were fixed by acetone (Gray et al., 2012 (link)). Sections were stained with the flowing antibodies: anti-Lyve1-A647, anti-CD3ε-bio (clone 145-2C11), Goat anti-CCL20 (AF760), Polyclonal Rabbit anti-GFP (Thermo Fisher Scientific), anti-B220-A647 (RA3-6B2), anti-ICAM1-bio (3E2), anti-IL7Rα-A647 (A7R34), rat anti-scart2, Hamster anti-TCRγδ (GL3), Donkey anti-Goat-biotin Jackson immunoresearch), Goat anti-Armenian Hamster-bio (Jackson immunoresearch), Donkey anti-Rat-bio (Jackson immunoresearch), Anti-biotin-A488 (Jackson immunoresearch), Anti-biotin-Cy3 (Jackson immunoresearch). Images were captured with a Zeiss AxioOberver Z1 inverted microscope.