Anti smooth muscle actin sma

Tissues, dissected and separated into upper thigh, lower thigh, and calf, were preserved in 10% formalin and embedded in paraffin. Standard procedures were used to deparaffinize, and fixed antigens were retrieved through incubation at 60°C with 20 μg/mL Proteinase K (Sigma‐Aldrich) for 20 minutes or immersion in boiling unmasking solution for 20 minutes. Slides were blocked in PBS with 2% serum and 0.4% Triton for 30 minutes, followed by use of the Avidin/Biotin Blocking Kit (Vector Laboratories). Primary antibody incubation was for 12 hours at 4°C with 2% serum and 0.4% Triton. Secondary antibody incubation was for 1 hour at 21°C with a biotinylated antibody in PBS/0.4% Triton. Staining was visualized by using an ABC Elite kit followed by DAB treatment (Vector Laboratories, Burlingame, CA) and hematoxylin and eosin counterstaining. Vessels were immunostained with anti‐CD31 (BD Biosciences Pharmingen) and anti–smooth muscle actin (SMA; Dako). The hVEGF antibody was from R&D Systems.