The in vitro and in vivo expression of the rat CD31, Flk-1, and vWF proteins was assessed by western blotting. Seven days after implantation, the samples were harvested and stored in liquid nitrogen until evaluation. The frozen samples were completely homogenized in RIPA lysis buffer (50 mM Tris-HCl, 0.1% Triton X-100, 2 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, and 1 mM protease inhibitors) for 30 min at 4 °C and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
After being transferred to a poly(vinylidene fluoride) membrane (Millipore, MA, USA), the membranes were incubated with a solution of 5% milk powder in Tris-buffered saline (TBS) to prevent nonspecific interactions. Then, the membranes were incubated overnight with the antibodies and detected using an ECL (ECL western blotting substrate, Pierce, USA) system. The specific primary antibodies used for this experiment were mouse anti-rat CD31 (Abcam, UK), rabbit anti-rat Flk-1 (Abcam, UK), and mouse anti-rat vWF (Santa Cruz, USA) antibodies.
After being transferred to a poly(vinylidene fluoride) membrane (Millipore, MA, USA), the membranes were incubated with a solution of 5% milk powder in Tris-buffered saline (TBS) to prevent nonspecific interactions. Then, the membranes were incubated overnight with the antibodies and detected using an ECL (ECL western blotting substrate, Pierce, USA) system. The specific primary antibodies used for this experiment were mouse anti-rat CD31 (Abcam, UK), rabbit anti-rat Flk-1 (Abcam, UK), and mouse anti-rat vWF (Santa Cruz, USA) antibodies.