Alexa 488 for green

The vascular grafts were explanted and fixed in 4% paraformaldehyde at 4°C. Cross sections in the middle portion of the graft (10 μm in thickness; 5–7 mm from the proximal end of the graft) were cryosectioned for H&E staining and immunostaining. The frozen sections (10 μm in thickness) of vascular grafts were incubated in 5% normal goat serum for 30 minutes to block the non-specific binding, and then incubated with primary antibody diluted in 5% normal goat serum overnight at 4°C. Negative controls were included by omitting the primary antibody. The sections were incubated for one hour with either horseradish peroxidase-conjugated anti-mouse or rabbit IgG (1:1000, Alexa 594 for red and Alexa 488 for green, Invitrogen, Carlsbad, CA). Finally, slides were mounted and examined by using a fluorescence microscope (Zeiss Axioskop 2 MOT). For immunohistochemical staining, the sections were incubated with biotin-conjugated secondary antibody and avidin-biotin enzyme reagent for 1 h. A hematoxylin and eosin (H&E) counterstaining was performed. For H&E staining, sections were deparaffinized first in three changes of 100% xylene for 5 minutes each, then hydrated through graded alcohol (100%, 95% and 70%; 5 min each), and rinsed in phosphate-buffered saline, followed by standard H&E staining.