Retinoic acid is required for <i>in vitro</i> differentiation of ovine tracheal epithelial cells.

<p>Ovine tracheal epithelial cells were cultured at ALI for 21 days with the indicated concentrations of retinoic acid. (A) Immunofluorescent staining with anti-β-tubulin (green), rhodamine-phalloidin (red) and DAPI (blue). (B) Scanning electron microscopy. (C) Immunofluorescent staining with anti-ZO-1 (green) and DAPI (blue). (D) Haematoxylin and eosin-stained histological sections. (E) Quantitation of ciliation as percentage of total area from β-tubulin staining. (F) Trans-epithelial electrical resistance measurements. Data shown are from a single representative animal with mean +/- standard deviation from three inserts displayed. (G) Cell layer thickness measured from three points per field in H&E-stained sections. (H) Cell layer thickness as determined by counting nuclei at three points per field in H&E-stained sections. (I) Quantitation of ciliation by counting ciliated cells in H&E-stained sections. (E, G, H, I) Five images from each of three inserts were analysed and data displayed is mean +/- standard deviation from four animals. Statistical significance was assessed by Student’s <i>t</i>-test (E, G, H and I) or one-way ANOVA with Dunnet’s post-test (F). Significance values are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01) or three (<i>P</i><0.001) asterisks. Black asterisks indicate all samples were significantly different to untreated control in panel F.