Identification of neutrophils in the airway wall. (a) Representative image of a microdissected whole-mount mouse conducting airway specimen stained for actin (SiRActin, light cyan) to visualize epithelial and smooth muscle cells and with anti-Ly6G/Gr-1 antibodies (green) to visualize neutrophils. Images from four segments (dotted line squares) were acquired as z-stacks for statistical analysis. Scale bar: 1000 μm. (b) Higher magnification images of one of the segments indicated in (a) showing a fragment of the airway wall: epithelium and smooth muscles layer (left panel), neutrophils (green, middle panel), and merged image (right panel). Grid spacing: 20 μm. (c) Higher magnification selective z-stack images of the region marked in (b) showing the epithelial layer (left image of z-stack), smooth muscle layer (right image of z-stack), and airway wall neutrophils (arrow, middle image of z-stack). The three-dimensional image shown in the far-right panel was generated by volume rendering of the x–y and y–z projections. An airway wall neutrophil is indicated with an arrow. Nuclei were stained with NucBlue (blue). Scale bar: 15 μm; grid spacing: 5 μm.