Figure 5

HS structure is abnormal in hypoplastic nitrofen treated rat lungs. HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.