Af3667 sp

Lung tissue samples were lysed in a buffer containing Tris–HCl, SDS, glycerol, and other reagents. The resulting lysate was then centrifuged and the protein concentration was determined using the BCA method. Equal amounts of protein were separated using SDS-PAGE and transferred to PVDF membranes. The membranes were blocked and incubated with primary antibodies against myeloperoxidase (MPO) (R&D: AF3667-SP; 0.5 µg/mL) and neutrophil elastase (NE) (CST: E8U3X; 1:1000), followed by a secondary antibody, and protein bands were detected using chemiluminescence. ImageJ software was used to calculate the ratio of MPO and NE to β-actin, and significance was determined by Student’s t test (P < 0.05) using GraphPad Prism software.

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)].