Bp0075

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)].

In vivo neutrophil depletion was performed by intraperitoneal (i.p.) injection of 500 μg sterile anti-Ly6G monoclonal antibody (Clone 1A8, BP0075, Bio X Cell) or control rat IgG (14131, Sigma) to both female and male C57BL/6 mice beginning one day prior to tumor inoculation and was continued to maintain depletion throughout the tumor study (days-1, 1, 4, 8, and 11). Depletion of neutrophils (Gr1^hi) in lungs was confirmed by flow cytometry at the time of sacrifice.
NK cell depletion was performed by i.p. injection of 200 μg sterile anti-NK1.1 mAb (Clone PK136, BE0036, Bio X Cell) or control mouse IgG (I5381, Sigma-Aldrich) to both female and male C57BL/6 mice beginning one day prior to tumor inoculation and was continued to maintain depletion throughout the tumor study (days-1, 1, 4, 8, and 11). Depletion of NK1.1 cells in lungs (NK1.1⁺CD49b⁺) was confirmed by flow cytometry at the time of sacrifice.