L6274

One drop of each sample of soluble protein was placed in a nitrocellulose membrane. After drying, membranes were incubated for 1 h with 5% BSA, with agitation, at room temperature. Subsequently, membranes were incubated with primary antibody:mouse antiCollagen type I 1:1000 (#ab90395, abcam, Cambridge, UK); rabbit antiCollagen type IV, 1:500 (#ab6311, abcam, Cambridge, UK); rabbit antifibronectin 1:500 (#ab45688, abcam, Cambridge, UK); and mouse antilaminin, 1:500 (#L8271, Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation, membranes were washed 3× for 5 min with Tris Buffered Saline (TBS) with Tween 20 and then the R.T.U. VECTASTAIN^® Universal ABC Elite^® Kit (#PK-7200, Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody, in accordance with manufacturer’s instructions. Finally, incubation was revealed using a Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories, Burlingame, CA, USA). Extraction buffer without samples was used as a negative control. Collagen type I (#sc-136157, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Collagen type IV (#C5533, Sigma-Aldrich), fibronectin (#FC010, Sigma-Aldrich, St. Louis, MO, USA) and laminin (#L6274, Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. At least three independent samples were used in each condition.

RGCs were isolated from postnatal day three Sprague-Dawley pups as described^{61 (link)} and seeded immediately in neurobasal-SATO (nb-SATO) media at 3,000 RGCs/well in 96-well plates (Falcon 087723B, Corning Inc, Corning, NY) coated with poly-D-lysine (70 kDa, 10 μg/ml, Sigma-Aldrich Corp., St. Louis, MO) and laminin (2 μg/ml, L-6274, Sigma-Aldrich Corp., St. Louis, MO). RGCs were cultured at 37 °C, 10% CO₂ for 3 days with MBV (0-80 µg/ml) or UBM-ECM (250 μg/ml). Using a Live/Dead Imaging Kit (R37601, Life Technologies, Carlsbad, CA), the number of viable RGCs per area was quantified^{14 (link)}. Five random, non-overlapping fields were imaged at 20×, totaling 45 fields of view. For total neurite growth, RGCs were labeled with anti-βIII tubulin (1:300, TUJ-1, MAB5564, Millipore, Burlington, MA). The first ten RGCs encountered, not contacting another RGC, were measured, totaling 90 RGCs per group, as described^{62 (link)}.

ECM proteins were used to coat tissue culture plastic using the recommended manufacturer's concentrations and a value above (high) and below (low) that concentration (medium). The ECM proteins used were collagen I from human neo-natal fibroblasts (Advanced Biomatrix, San Diego, USA) at 38.7 µg·mL⁻¹, 387 µg·mL⁻¹ and 968 µg·mL⁻¹; collagen IV from human placenta (C8374, Sigma-Aldrich, Dorset, UK) at 10 µg·mL⁻¹, 250 µg·mL⁻¹ and 500 µg·mL⁻¹; vitronectin from human plasma (V8379, Sigma-Aldrich) at 0.5 µg·mL⁻¹, 1.25 µg·mL⁻¹ and 2.5 µg·mL⁻¹; laminin from human placenta (L6274, Sigma-Aldrich) at 1 µg·mL⁻¹, 2 µg·mL⁻¹ and 4 µg·mL⁻¹; and 0.1% fibronectin from human plasma (F0895, Sigma-Aldrich) at 1 µg·mL⁻¹, 25 µg·mL⁻¹ and 250 µg·mL⁻¹. Non-adherent 96-well plates (10 554 961, Fisher Scientific, Hemel Hempstead, UK) were coated with ECM proteins following the relevant manufacturer's protocol.