Ab5392

Before microscopic examination, the neuron cultures (in the microfluidic device) were fixed for 30 min in 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized in 0.25% Triton X-100 for 30 min and then blocked with 4% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The cultures were incubated with primary antibodies (diluted in 1% casein) for 4 h at room temperature and then with secondary antibodies for 2 h. All excessive antibodies were removed by rinsing with PBS after each incubation period. The primary antibodies included mouse anti-βIII tubulin (R&D, MAB1195; 1:500), rabbit anti-GFP (Thermo Fisher, A21311; 1:500), chicken anti-MAP2 (Abcam, AB5392; 1:2000), and mouse anti-Tau-1 (Millipore, MAB3420; 1:500).

Single IHC labeling was performed according to previously published protocols. Briefly, tissue sections were dewaxed in xylenes, rehydrated to water via graded ethanols, and immersed in 3% aqueous hydrogen peroxide for 15 min to quench endogenous peroxidase activity. Antigen retrieval was achieved via microwave pressure cooker at high power for 8 min, submerged in Tris EDTA buffer (pH 8.0). Sections were then incubated overnight at 4°C using antibodies specific for MAP2 (Abcam, Cat# ab5392 Lot# RRID: AB_2138153, 1:1000), parvalbumin (Millipore Cat# MAB1572 Lot# RRID: AB_2174013, 1:1000), and the N-terminal amino acids 66–81 of the amyloid precursor protein (APP; Millipore, Cat# MAB348 Lot# RRID: AB_94882, 1:80K). Slides were then rinsed and incubated in the relevant species-specific biotinylated universal secondary antibody for 30 min at room temperature. Next, application of an avidin biotin complex (Vector Laboratories, Cat# PK-6200 Lot# RRID: AB_2336826) was performed for 30 min, also at room temperature. Lastly, the 3,3′-diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Cat# SK-4100 Lot# RRID: AB_233638) was applied according to manufacturer’s instructions. All sections were counterstained with hematoxylin, dehydrated in graded ethanols, cleared in xylenes, and coverslipped using cytoseal-60.

Neuron-microglia co-cultures were quantified by immunofluorescent staining using antibodies for MAP2 (1:1000, Abcam, ab5392), synaptophysin (1:2000, Sigma, S5768), PSD95 (1:250-1:500, Abcam, ab2723), and Iba-1 (1:500, Wako, 019-19741) respectively. Microglia cells were centered for image capture by Leica TCS-SP8 Laser Scanning microscope with ×40 objective. To quantify the density of synaptophysin or PSD95 puncta, concentric circles were drawn around the microglia at increments of 20 μm. The puncta number within each concentric circle were counted by Sholl analysis. Synaptic density was calculated by puncta number/neurites length in the given area. All images were processes using ImageJ. Researchers were blinded to genotype during imaging.