Fitc conjugated anti f4 80

Peritoneal cells were harvested from BALB/c mice. Analyses of apoptosis were performed as described elsewhere (34 (link)). The cells were then plated in 24-well plates at a density of 1 × 10⁶ cells per well. If necessary, SNA (10 μg/ml) pretreatment was applied. A/WSN/1933 virus or UV-WSN virus was added to each well at a 1 × 10⁶ pfu. After incubation at 37°C for different time periods, cells were harvested and stained with anti-FcγRII/III (Fc receptor blocker) before being stained with APCef780-conjugated anti-CD19 (Catalog No: 47-0193-80, eBioscience), PE-conjugated anti-CD23 (Catalog No: 12-0232-81, eBioscience), FITC-conjugated anti-B220 (Catalog No: 553088, BD Biosciences), FITC-conjugated anti-F4/80 (eBioscience), and BV421-conjugated anti-CD11b antibodies (Catalog No: 48-0112-80; eBioscience). After 1 h incubation at 4 °C, cells were washed with FACS buffer and stained with APC-conjugated Annexin V (Catalog No: 17-8007-74, eBioscience) for 15 min at room temperature. To exclude dead cells, stained cells were incubated with 10 μg/mL of PI (eBioscience, San Diego, CA, USA) or 7AAD (Catalog No: 51-68981E, BD Biosciences). The cells were analyzed with a FACSCanto^TM II.

Paraffin sections of lung were deparaffinized in xylene and hydrated through graded alcohols. Sections were incubated overnight at 4°C with anti-IL-33 antibody or anti-normal goat IgG as the control (R&D Systems Inc., Minneapolis, MN, USA). Streptavidin-biotin amplification (Dako, Denmark) was carried out for 30 minutes, and hematoxylin was used to counterstain. For immunofluorescence, sections were incubated overnight at 4°C with anti-IL-33 antibody, and Northern Light 557 conjugated anti-goat IgG (R&D Systems Inc.) was used as the secondary antibody. FITC-conjugated anti-F4/80 was used to identify tissue macrophages (eBioscience, San Diego, CA). Fluorescent images were visualized using a bio-imaging navigator microscope system (Olympus, Tokyo, Japan).