Metamorph advanced imaging acquisition software version 7

After the appropriate treatment, cells on coverslips were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and incubated with the appropriate primary antibody overnight at 4 °C. Cells were washed with PBS to remove excess primary antibody and incubated with the secondary antibody for 2 h at room temperature. Nuclei were stained with DAPI for 2 min. After mounting, fluorescence images were acquired and analyzed using the TCS SP8 confocal microscope (Leica, Wetzlar, Germany) and MetaMorph Advanced Imaging acquisition software version 7.8 (Molecular Devices, San Jose, CA, USA).

Lungs were collected from mice infected with Mabs-R for 21 days. The tissues were fixed in 10% formalin and embedded in paraffin wax. The sectioned tissues (4 μm) were stained with hematoxylin and eosin (H&E). H&E-stained images were scanned using the PANNORAMIC 300 Flash DX (3DHISTECH, Budapest, Hungary). To quantify the percentage of inflamed area, the red-stained area with high threshold from three lungs retrieved from sacrificed mice was measured and divided by each lung area using FIJI software. For immunohistochemical (IHC) staining, lung paraffin blocks were sectioned and stained with antibody specific for anti-mouse Ly6G (Bio X Cell, Lebanon, NH, United States, BP0075-1). For analysis of neutrophils, anti-Ly6G-stained lung tissue images were taken with a confocal laser scanning microscope. The relative integrated intensity of Ly6G-stained cells was measured from random lung tissue images using MetaMorph Advanced Imaging acquisition software version 7.8 (Molecular Devices, San Jose, CA, United States).