A1 rsi

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).

Protein expression was visualized using immunofluorescent (IF) staining after 14 days in culture. Cells were fixed in 4% paraformaldehyde for 45 min, permeabilized in 0.25% Triton X-100 for 15 min, and blocked in 3.5% w/v bovine serum albumin (BSA) at 4 °C overnight. Primary antibodies were diluted in PBS with 1% w/v BSA and 0.05% NaN₃ and were placed on samples overnight at 4 °C. Proteins visualized via IF were αSMA (abcam ab7817; 1:50), CD44 (Calbiochem 217594; 1:120), and RHAMM (Novus NBP1–95379; 1:120). Gels were washed 4× to remove unbound primary antibody over an 8 h period in PBS with 0.01% v/v Tween 20. Secondary antibodies (AlexaFluor 488/555/633, Invitrogen Carlsbad, CA) were added at 1:200 concentration overnight at 4 °C. Samples were counterstained with DAPI and AlexaFluor 488 or 633 conjugated phalloidin (Invitrogen) for 2 h prior to imaging with a confocal microscope (Nikon A1-Rsi or Zeiss LSM 510).