5XFAD mice exhibit intraneuronal Aβ42 prior to amyloid deposition and neuron loss. Parasagittal brain sections from 1.5 (G), 2 (H), 4 (A, I-T), 6 (B), 9 (C, E), and 12 (D, F) month mice were incubated with anti-Aβ42 C-terminal neo-epitope antibody, visualized by DAB staining, and micrographed. 5XFAD mice display progressive Aβ42-positive plaque deposition with age (A-D). In 5XFAD Layer 5 cortex, intraneuronal Aβ42 (arrows) is first observed at 1.5 months (G) and amyloid plaques at 2 months (H) of age. At high magnification, intraneuronal Aβ42 staining is confined within numerous small spherical puncta dispersed throughout the cytoplasm of the soma of large pyramidal neurons (K-O). The appearance of these Aβ42-positive puncta suggests membrane-bound intracellular compartments such as endosomes or lysosomes. Importantly, the absence of Aβ42 staining in 5XFAD; BACE1−/− neurons (E, P-T) demonstrates the selectivity of the antibody for Aβ42, and indicates that the antibody dos not cross-react with endogenous or transgenic APP under the conditions used. Aβ42 staining is also lacking in non-transgenic littermate (Non-Tg) sections (F, J). Scale bar = 400 μm (A-F), 40 μm (G-J), 9 μm (K-T).