Figure 6. Infected region of the mammary gland from a US dairy cow infected with highly pathogenic avian influenza A(H5N1) virus, labeled with IAV-Np (yellow pseudocolor, DyLight 594), individually duplexed either with epithelial marker pan-cytokeratin (red pseudocolor, Alexa Fluor 647) (B, C) or macrophage or monocytic marker ionized calcium binding adaptor molecule 1 (Iba1) (red pseudocolor, Alexa Fluor 647) (D) using fluorescent labeling. A representative image from a hematoxylin and eosin stain section highlights the cellular architecture of the affected mammary gland (A). Unaffected secretory alveoli were observed in the section (solid black outline). Secretory alveoli variably contain eosinophilic proteinaceous material and corpora amylacea. The secretory alveoli were lined by cuboidal epithelial cells (green arrows) that were variably vacuolated. A few secretory alveoli were disrupted by inflammation (macrophages and neutrophils) and epithelial necrosis (dashed black outline). A single interlobular duct (black asterisk) lined by a bilayer of low cuboidal epithelial cells (black arrows) was observed. The duct contains eosinophilic proteinaceous fluids with scattered inflammatory cells (macrophages and neutrophils) and sloughed epithelium (A). Epithelial cells lining secretory alveoli (B) and interlobular ducts (C) were labeled with pancytokeratin as expected. IAV-Np intranuclear co-labeling (white arrows) in epithelial cells lining the secretory alveoli (B) and ducts (C). Intraluminal cells within secretory alveoli had intranuclear IAV-Np labeling (yellow arrows) that variably co-labeled with pan-cytokeratin. Likewise, Iba1 labeling (green arrows) was observed in the lumens of secretory alveoli or interlobular (alveolus labeling highlighted by white dotted outline) and interstitium (highlighted by the solid white outline) (D). Iba1 labeling was intense, diffuse, and cytoplasmic. IAV-Np intranuclear and intracytoplasmic labeling was less commonly co-labeled within Iba1 labeled cells (white arrows) (D). IAV-Np labeling was not observed in Iba1-labeled cells in the interstitium solid white outline). Insets highlight intranuclear labeling in panels C, B, and D, and the white boxes in the corresponding images represent the origin of the inset image. Scale bars indicate 200 μm (A), 50 μm (B, C, D), and 20 μm (insets). IAV-Np, influenza A virus nucleoprotein; Iba1, ionized calcium binding adaptor molecule 1.