Cy5 or cy3 coupled secondary antibodies

Femoral artery cross-sections were incubated with antibodies recognizing α-smooth muscle actin (α-SMA, Sigma-Aldrich), calponin (Abcam, Cambridge, UK), Ki-67 (Abcam), von Willebrand factor (vWF, Dako, Glostup, Denmark), monocyte + macrophage antibody (MOMA)-2 (Serotec, Oxford, UK), Sca-1/Ly-6A/E (R&D Systems, Minneapolis, MN, USA), ICAM-1 and VCAM-1 (Santa Cruz Biotechnology, Dallas, TX, USA), and CD34 (BD Pharmingen, Franklin Lakes, NJ, USA). Ensuing incubations were carried out with Cy5-or Cy3-coupled secondary antibodies (Molecular Probes, Eugene, OR, USA) and counterstained with nuclear 4.6-diamidino-2phenylindole (DAPI) (Linares, Wertheim, Germany). Monoclonal antibodies to α-SMA were labeled directly with Cy3. For negative controls, the primary antibody was substituted by an appropriate species-and isotype-matched control antibody (Santa Cruz Biotechnology). Function-blocking mouse anti-human ICAM-1 (cloneP2A4) and VCAM-1 (clone P1B8) antibodies were obtained from Chemicon International (Hempshire, UK). Semi-quantitative analysis of immunohistochemistry was performed using a visual scale ranging from 1 to 4, indicating very low staining for 1 and very strong staining for 4.