Journal: BMC Developmental Biology
Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?
Figure Lengend Snippet: Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.
Article Snippet: Bound antibody was detected with rabbit VSV-G tag antibody (Abcam, Cambridge, UK), diluted 1/200 in 1% (v/v) goat serum in PBS, for 2 h at room temperature, followed by FITC conjugated goat anti-rabbit IgG (Sigma-Aldrich, Gillingham, UK), diluted 1/500 in the dark for 1 h. Controls were the omission of HS antibody or treatment of sections with heparitinase (EC 22.214.171.124) (IBEX Technologies Inc, Canada) overnight at 37°C (changing enzyme after 4 h), prior to antibody incubation, to remove HS epitopes.
Techniques: Staining, Immunohistochemistry