Figure 1.

Histological assessment of BBEC differentiation over time. Bronchial epithelial cell cultures were grown for the indicated number of days at an ALI (relative to the establishment of the ALI), fixed and paraffin embedded using standard histological techniques. Samples of ex vivo bronchial epithelial tissue were similarly fixed and embedded. Sections were cut, deparaffinised and stained using (A) H&E and (B) immunohistochemistry with an anti-p63 antibody labelling the nuclei of basal cells (brown staining). Representative images are shown of ex vivo bronchial epithelium and BBECs grown for 3, 12, 21 and 42 days post-ALI (see also Figs S1 and S2). Quantitative analysis (using ImageJ) was performed of histological sections of BBEC layers fixed at three-day intervals ranging from day −3 to day 42 post-ALI (see Fig. S1); the epithelial thickness (C) and number of cell layers (D) comprising the epithelium were measured. For each insert, three measurements were taken (left, centre and right) in each of five randomised 400x fields of view evenly distributed across the strand; three inserts were analysed per time-point and the data represents the mean +/−standard deviation from tissue derived from three different animals (n = 9). Ordinary One-way ANOVA statistical analyses with post-test for linear trend demonstrated significant (P ≤ 0.05) increasing trends over time for both epithelial thickness (C) and the number of cell layers (D).