Bz x800 digital microscope

The D-17 and A-72 cells (1 × 10⁵ cells/sample) were fixed with 4% paraformaldehyde in PBS for 10 min and quenched with 50 mM NH₄Cl in PBS containing 0.2 mM Ca²⁺ and 2 mM Mg²⁺ (PBSc/m) for 10 min. The cells were treated with blocking buffer (PBSc/m supplemented with 0.5% BSA) for 30 min and incubated with 10 µg/mL of E134Bf or the control blocking buffer for 1 h. The cells were then incubated with Alexa Fluor 488-conjugated anti-dog IgG (1:400; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and DAPI (Thermo Fisher Scientific Inc.) for 45 min. Fluorescence images were obtained using a BZ-X800 digital microscope (Keyence, Osaka, Japan) at a magnification of 40× with a GFP filter channel (green) and a DAPI filter channel (blue). All experiments were conducted in triplicate.

Mice were euthanized and the lungs were perfused via the right ventricle with 3 mL of PBS. Using a catheter (26751, Exel International) inserted into the trachea, the lungs were inflated with 1 mL of 10% formalin solution, removed, and subsequently submerged in 10% formalin solution at room temperature for 24 h. The lungs were then transferred into 70% ethanol and subjected to paraffin embedding, sectioning, and hematoxylin and eosin (H&E), Alcian blue periodic acid-Schiff (ABPAS), or trichrome staining by the Comparative Pathology Laboratory at UAB. Lung sections were also subjected to immunohistochemical (IHC) staining with IL1B (AF-401-NA, R&D Systems) or CXCL2 (701126, Thermo Scientific) using a mouse and rabbit HRP/DAB detection kit (ab64264, abcam). Images were captured on a Nikon Ts2-FL inverted microscope with a Digital Sight 10 camera or a Keyence BZ-X800 digital microscope.
For quantification of bronchial epithelial thickness and goblet cell size, randomly selected regions of each ABPAS-stained slide were chosen for measurement and analyzed as previously described^{31 (link),32 (link)}. Briefly, bronchial epithelial cells were measured from their base to their highest point in the lumen. Goblet cell size was quantified by calculating the area of randomly selected goblet cells and normalized to the luminal area of the corresponding airway from each animal.