F actin

All ECM samples were fixed in 4% paraformaldehyde for 20 min, washed three times each with 0.01% Triton X-100 in phosphate buffered saline (PBS, pH 7.4) and PBS, blocked with 3% fish gelatin, and immunolabelled with anti-collagen IV (catalog # ab6311, Abcam, Cambridge, MA, USA), anti-laminin (catalog # ab11575, Abcam, Cambridge, MA, USA) or anti-Fibronectin (catalog # ab6584, Abcam, Cambridge, MA, USA) followed by species appropriate fluorophore-tagged secondary antibody. Where appropriate, samples were stained with F-actin (catalog # 00045, CF594 Phalloidin, Biotium, Fremont, CA, USA) to confirm decellularization, and nuclei were counterstained with DAPI (catalog # D1306, FisherScientific, CA, USA). All experiments were performed for all cell strains, with at least 3 samples per cell strain. For each immunolabelled sample, 3–8 random locations were imaged using a Leica DMi8 inverted fluorescence microscope. Average fluorescence intensity of immunolabelled samples were quantified and semi-quantitative analyses is shown here. As appropriate, representative images from a single field of view for a single donor are illustrated.