Glial activation is increased in the EFAD subiculum and cortex, while E4FAD mice exhibit elevated IL-1β levels. Astrocytes and microglia were visualized in sagittal brain sections of 6-month-old EFAD mice using immunohistochemistry. Initial DAB staining revealed prominent gliosis in two regions of the brain: the subiculum and deep layers of the cortex. (A) Extensive GFAP-immunoperoxidase staining was evident in the subiculum of all EFAD mice. In addition, dystrophic astrocytes could be seen throughout the deep layers, but not in superficial layers, of the EFAD cortex. Insets: 20× magnification of the subiculum (dashed red box). Magenta arrows: Clusters of dystrophic astrocytes in the cortex. Scale bar: 500 μm. (B) Activated microglial cells were clearly visible in the subiculum of all EFAD mice and were present in deep cortical layers as well. E4FAD sections exhibited more activated microglia in the deep cortex than E2FAD and E3FAD sections. Green arrows: Activated microglia. Scale bar: 500 μm. (C) Double immunofluorescence staining for GFAP (magenta) and Iba1 (green) confirmed region-specific glial activation in EFAD brains. Representative images of EFAD subicula are shown. Scale bar: 30 μm. (D) Levels of the pro-inflammatory cytokine IL-1β were measured using ELISA in 6-month-old EFAD cortex (CTX) extracts. E4FAD sections exhibited significantly higher levels of IL-1β than E3FAD sections. One-way ANOVA, ** P < 0.01.