Confocal microscopy analysis of Aβ-associated microglial activation in EFAD mice. Sagittal brain sections from 6-month-old EFAD mice were analyzed for Aβ deposition and associated microglial activation in two regions of interest, the subiculum and deep layers of the cortex. Reactive microglial cells within the vicinity of Aβ plaques consistently exhibited an amoeboid-like shape with dystrophic processes. (A) Two-dimensional projection image (approximately 20 μm, z-axis) of a E3FAD subiculum double stained for Aβ1–42 (MOAB2) and activated microglia (Iba1). Image overlays depict metrics used to characterize and quantify Aβ deposition and associated microglial activation. White arrows: Examples of plaque domains. (B) Grayscale image of Aβ plaques chosen for analysis. Plaque types, size of plaques and MOAB2 fluorescence intensity were quantified. Magenta: Traced plaques. (C) Grayscale image of activated microglia with plaque domain overlays. The number of microglia cell bodies within plaque domains were collected and quantified along with Iba1 fluorescence intensity. Yellow ring: Plaque domains (4.42 mm2). (D) Thresholding was performed within plaque domains to quantify the percentage area occupied by microglia cell bodies and processes. Representative minimum threshold value of 35 (0 to 255 brightness scale) depicted. Blue: Background. Scale bars: 20 μm.