Celena s digital fluorescence imaging system

To verify the cellular source of IL-22 in NP tissues, immunofluorescence analysis was conducted using primary antibodies directed against anti-IL-22 (1:100; Abcam, Cambridge, MA, USA), anti-mast-cell tryptase (1:500; Abcam), anti-eosinophil peroxidase (EPX) (1:200; Abcam), and anti-human neutrophil elastase (HNE) (1:500; Abcam). After 24 hours of incubation of primary antibodies at 4°C, the secondary antibody Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1,000; Abcam) or Cy3-conjugated goat anti-rabbit (1:500; Abcam) was incubated for 1 hour at room temperature. Then, the nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1,000; Sigma-Aldrich) for 2 minutes. The tissues were mounted with Fluoroshield Mounting Medium (ab104135; Abcam). Fluorescent images were obtained using the CELENA^® S Digital Fluorescence Imaging System (Logos Biosystems, Annandale, VA, USA). The proportion of IL-22-positive cells on each of the tryptase, EPX, and HNE-positive cells was calculated and analyzed in 3 randomly selected fields, and non-specific signals were excluded.