Immunohistochemistry, imaging and quantification

Immunohistochemical procedures were performed as detailed previously^85,86 for mouse anti-human Ki67 (1:100, BD Biosciences), rabbit anti-GFAP (Dako Diagnostic, 1:1000), and guinea-pig anti-DCX (Millipore, 1:3000). For rabbit anti-Iba-1 (Wako, 1:400) and mouse anti-post synaptic density-95 (Millipore, 1:160) the sections were blocked overnight in 10% normal donkey serum/0.2% triton x at 4 °C followed by overnight incubation in the primary antibodies at 4 °C. Fluorescence primary antibodies were detected using species-appropriate Alexa Fluor conjugate secondary antibodies, Alexa 488 Donkey anti-rabbit (H + L) (A21206, Invitrogen, 1:1000), Alexa 555 Goat anti-guinea pig (H + L) (A21435, Invitrogen, 1:1000), Alexa 647 Donkey anti-mouse (H + L) ({"type":"entrez-protein","attrs":{"text":"A31570","term_id":"85652","term_text":"pir||A31570"}}A31570, Invitrogen, 1:1000). For brightfield immunohistochemistry, rabbit anti-Abeta (37–42, NEB, 1:100) and mouse anti-Abeta 1–16 (Covance, 6E10, 1:200) primary antibodies were detected using species-appropriate biotinylated secondary antibodies biotinylated goat anti-mouse (H + L) (115-065-146, Jackson Immuno Research, 1:1000) or biotinylated goat anti-rabbit (H + L) (111-065-144, Jackson Immuno Research, 1:1000), and the signal was amplified using the avidin–biotin–peroxidase system (VectaStain ABC Kit, Vector Laboratories) and revealed using a 3,3-diaminobenzidine (DAB)-containing solution (Sigma-Aldrich).

Ki67, GFAP, and DCX was imaged on a motorized Olympus IX81 microscope. For quantification of neurogenesis markers expressed by SVZ and dentate gyrus cell populations, 4–6 sections at 120 µm intervals through the striatal SVZ or dentate gyrus were quantified. Counts were limited to the subventricular zone and subgranular zone respectively. All Abeta and Tau sections were scanned with a 20 × 0.75 NA objective with a resolution of 0.3225 µm (BX61VS, Olympus, Toronto, Ontario). Quantification of amyloid beta Abeta (37–42) and Abeta (1–16) was performed in Fiji v1.52p⁸⁴ using the process and analyze particle functions to obtain arbitrary units (arb. units) per animal. For colocalization of PSD95 and Iba-1, sections were imaged on a confocal Leica SP5 using ×63 oil 1.4 NA objective with 1× zoom and 2048 × 2048 resolution, 400 Hz. 20–30 µm z-stacks were acquired at 0.4 µm z-step size, using sequential scans with 2× frame averaging and 2× line averaging for both the 488 nm (Iba-1) and 647 nm (PSD95) wavelengths. For quantitation, image brightness was adjusted systematically to all stacks and 3D rendering of confocal images was performed using 3D project in Fiji (v1.52p). Only puncta larger than approximately 0.4 µm were counted using the manual cell counter plug-in. Iba-1+ microglial cells in CA1 with complete arborization were quantified, WT-D (n = 46 from 5 animals), 3xTg-D (n = 40 cells from 4 animals), WT-S (n = 42 cells from 5 animals), 3xTg-S (n = 43 cells from 5 animals). The experimenter was blinded to the genotype and treatment during image acquisition, processing and quantification of all IHC experiments.