Quantifying Hippocampal Microglia via IHC and Stereology

Immunohistochemistry (IHC) and stereological analysis of F4/80+ cells in the dorsal hippocampus were performed as described previously [20 (link)]. Briefly, 9-month-old mice were perfused transcardially with PBS and immersion-fixed in 4% formaldehyde overnight. Following transfer to a sucrose gradient, 40 μm sections were cut using a cryostat. IHC was performed on free-floating sections using a monoclonal rat anti-mouse F4/80 primary antibody (1:10,000; Serotec, Raleigh, NC, USA) and a biotinylated goat anti-rat immunoglobulin G (IgG) secondary antibody (1:3000; Vector Laboratories, Inc., Burlingame, CA, USA), after which sections were treated with avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories, Inc., Burlington, CA, USA). Staining was visualized using diaminobenzidine (DAB; Vector Laboratories) and counterstained with hematoxylin (Fisher Scientific, Fair Lawn, NJ, USA). Stereological counting was performed using a Nikon 218912 light microscope interfaced with the StereoInvestigator software package (MicroBrightField, Williston, VT, USA). IgG leakage was assessed in adjacent sections (5 per mouse) as above except we used a biotinylated anti-mouse IgG antibody (1:3000).