Mouse collagen type 4

Plates were coated with a solution of rat plasma FN or of the 70-kDa fragment at 0.5 and 2.0 μg/ml in CAPS buffer (10 mM N-cyclohexyl-3-aminopropanesulfonic acid [CAPS], pH 11, 150 mM NaCl) in triplicate by incubating overnight at 4°C. Wells were blocked with 1% BSA in PBS and then incubated with 30 μg/ml mouse type IV collagen (BD Biosciences) for 1 h with or without 100 μg/ml gelatin (Sigma-Aldrich). Bound collagen was detected by enzyme-linked immunosorbent assay (ELISA) with anti-collagen IV antibody and biotinylated goat anti-rabbit IgG (Pierce) at room temperature. Bound antibodies were detected with streptavidin β-galactosidase (Life Technologies) diluted 1:500 in 1% BSA with 1.5 mM MgCl₂ and 2 mM β-mercaptoethanol for 1 h at 4°C, followed by nitrophenyl β-d-galactopyranoside (Sigma-Aldrich) at 1 mg/ml in substrate buffer (50 mM Na₃PO₄, pH 7.2, 1.5 mM MgCl₂) for 50 min at room temperature. The reaction was stopped with 0.5 M Na₂CO₃, and absorbance at 405 nm was measured using an iMark Microplate Reader (Bio-Rad).