Cells cultured on all hydrogel scaffold combinations were fixed in 4% paraformaldehyde for 45 minutes and permeabilized with 0.25% Triton-X for 15 minutes at room temperature. Next, samples were the blocked in PBS with 3.5% bovine serum albumin (BSA) at 4°C overnight. The samples were incubated with primary antibodies of interest at manufacturer recommended dilutions in PBS and 0.35% BSA overnight at 4°C. Negative controls were left incubating in PBS with 0.35% BSA. The next day, samples were incubated with fluorescent secondary antibodies (AlexaFluor 488/555/633; Pacific orange, Invitrogen) overnight at 4°C. After washing, samples were counterstained with DAPI and phalloidin for 1 hour prior to imaging with confocal microscopy (Nikon A1-Rsi or Zeiss LSM 510).
Antigens investigated in this study included the endothelial marker CD31 (Abcam, Cambridge, MA., ab28364, 1:50); VIC activation marker αSMA (Abcam, ab7817, 1:50); eNOS (BD biosciences, BD610296, 1:100); and extracellular proteins laminin (Lam, Abcam, ab14055, 1:200), collagen type IV (Col IV, Abcam ab6586, 1:500), perlecan (Pln, Abcam, ab26265, 1:1000), collagen type I (Col I, Abcam, ab34710, 1:50) and fibronectin (FN, abcam, ab6328, 1:100).
Antigens investigated in this study included the endothelial marker CD31 (Abcam, Cambridge, MA., ab28364, 1:50); VIC activation marker αSMA (Abcam, ab7817, 1:50); eNOS (BD biosciences, BD610296, 1:100); and extracellular proteins laminin (Lam, Abcam, ab14055, 1:200), collagen type IV (Col IV, Abcam ab6586, 1:500), perlecan (Pln, Abcam, ab26265, 1:1000), collagen type I (Col I, Abcam, ab34710, 1:50) and fibronectin (FN, abcam, ab6328, 1:100).