Fibronectin

Gels are placed in a multiwell dish inside the sterile hood and UV sterilized for 15 minutes (Figure 1c). Once the sterilization is completed, 1.5mL of a fibronectin (human plasma, Corning) solution in 1X PBS (25 μg/mL) is pipetted inside each well. The gels are put at 37°C inside an incubator overnight. The following day, gels are extensively washed with sterile 1X PBS to remove any excess of fibronectin and residual monomers. Laminin (EHS murine sarcoma, Sigma-Aldrich) coating is performed dispensing a drop of a 25 μg/mL laminin solution on a sterile Parafilm sheet and placing the gels upside down on top to ensure the complete coverage of the surface. Fluorescence imaging of the fibronectin coating on PAA-OH gels is obtained by adding fluorescent fibrinogen to the coating solution (2 μg/mL AF488 labelled fibrinogen, Thermo Scientific). Laminin coating is visualized by indirect immunofluorescence staining: using a primary anti-laminin antibody (Invitrogen, PA1-16730), diluted 1:50 and a AF488-conjugated secondary antibody (Molecular Probes) diluted 1:200. Images are collected right after the drying step for fibronectin and after the immunofluorescence for Laminin using a Leica Stellaris confocal microscope equipped with a 10x air objective.