Dc 17

Total proteins were extracted from cultured bEnd.3 cells and brain tissues using protein lysis buffer (P0013C, Beyotime), containing 1% phosphatase inhibitors (Sigma-Aldrich), and 1% protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 12,000 × g (30 min, 4 °C). The total protein concentration was determined using a BCA protein assay kit (Beyotime, P0012) and was adjusted to 3.5 mg/mL. A total of 30 μg of protein was separated by 4–10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with Tris-buffered saline with Tween-20 (TBST) containing 5% milk and analyzed using monoclonal antibodies against Ki67 (1:2,000, SolA15; eBioscience), Cdk5 (1:2,000, DC 17; Santa Cruz), p-Cdk5 (1:2,000, C-7; Santa Cruz), mouse anti-glutamate receptor 1 (1:2000; ab183797, Abcam), mouse anti-synaptophysin (1:2000; S5768, Sigma-Aldrich), mouse anti-PSD95 (1:2000; ab13552, Abcam), total Tau (D1M9X, 1:2000, 46687, Cell Signaling Technology), phospho-Tau Ser^199/202 (1:2000, AB9674; Sigma-Aldrich) and rabbit anti-β-actin (1:2,000, 4970; Cell Signaling Technology). Blots were scanned using Image Quant Las4000mini System. ImageJ software (NIH) was used for the mean intensity of bands.

The following primary antibodies were used: mouse anti-Aβ_1-42 (1:1,000; A5213, Sigma-Aldrich), rat anti-CD68 (1:400; MCA1957, Bio-Rad), rabbit anti-Iba-1 (1:1,000; Wako Chemical), mouse anti-glutamate receptor 1 (1:400; ab183797, Abcam), mouse anti-synaptophysin (1:200; S5768, Sigma-Aldrich), rat anti-mouse Ly6G (1:200; BP0075, Bioxcell), mouse anti-myeloperoxidase (MPO, 1:1,000; AF3667-SP, R&D), mouse anti neutrophil elastase (NE, 1:1 000; MAB4517-SP, R&D), rat anti-Ki67 (1:500; SolA15, eBioscience), rabbit anti-Claudin5 polyclonal antibody (1:400; YT0953, Immunoway) mouse anti-pCdk5 (1:400; C-7, Santa Cruz) and mouse anti-Cdk5 (1:400; DC 17, Santa Cruz). The following secondary antibodies were used: Alexa Fluor 647 donkey anti-mouse, Alexa Fluor 594 donkey anti-rat, Alexa Fluor 488 donkey anti-goat, Alexa Fluor 555 goat anti-rabbit, and Alexa Fluor 488 goat anti-rabbit (1:400, Invitrogen). PE-conjugated anti-CD31 (1:100; 553373, B&D), FITC-conjugated anti-CXCL1 (1:100; IC4532G, B&D) and DyLight 649-labeled lectin (1:200; L32472, Invitrogen^TM) antibodies were used in some immunofluorescence experiments. The detailed immunofluorescence protocols have been described previously [30 (link)].