Fig. 3.

α₂-integrin immunostaining in CCAM and BPS. A–F: representative CCAM tissue sections stained for α₂-integrin with blue alkaline phosphatase immunohistochemistry and nuclear Fast Red counterstain. G–I: α₂-integrin stained with purple VIP-HRP immunolocalization and Hoxb5 protein (H, I) immunolocalized with brown DAB-HRP immunolocalization and counterstained with methyl green. Bar = 100 μm in D, 50 μm in A and E, and 25 μm in B, C, F, G, H, and I. A: α₂-integrin antibody 1, immunolocalized α₂-integrin protein to mesenchymal and epithelial cells of CCAM cysts (* in A and B) and adjacent compressed lung (arrow in A and C) with similar intensity. D, E, F: in contrast, α₂-integrin antibody 2 immunostaining of adjacent section of CCAM shown in A–C revealed different regional localization of α₂-integrin. Compared with adjacent compressed lung regions (# in D, E, F), increased α₂-integrin staining in mesenchyme and epithelial cells was seen around CCAM cyst (* in D, E, F). Using antibody 2, BPS tissue (G) showed intense purple epithelial (arrow in G) and mesenchymal localization (* in G) of α₂-integrin compared with human fetal lung (17-wk gestation) (H), which had strong but less intense purple α₂-integrin localization in epithelial cells of branching airway tips (long arrows in H). These α₂-positive purple epithelial cells were surrounded by closely adjacent subepithelial fibroblasts with intense brown nuclear staining for HoxB5 protein (arrowheads in H). Regions of airways with less purple α₂-integrin staining (< in H) are not surrounded by closely adjacent HoxB5-positive brown cells. α₂-integrin was also localized diffusely in mesenchyme, especially in fibroblasts with brown nuclear Hoxb5 staining (short arrow in H). In absence of α₂-integrin antibody, there is no purple α₂-integrin immunostaining (I) but continued Hoxb5 brown nuclear staining in presence of Hoxb5 antibody.