Ab11024

Vein samples were embedded in Frozen Section Medium (Richard-Allan Scientific Neg 50, Thermo Scientific) and frozen in dry ice-cooled isopenthane. Frozen tissue blocks were stored at —80C. The samples were cut in 8 um thick sections on a CryoStar™ NX70 Cryostat (Thermo Scientific™), mounted on SuperFrost Plus Adhesion slides and immediately post-fixed for 30 s in cold 95% ethanol. Slides were stored at —20C awaiting immunostaining.
Sections were immunostained for the presence of CD31 (1:50; Abcam #ab9498), von Willebrand factor (1:500; Abcam #ab6994), VEGFR-2 (1:200; Acris Antibodies TA337222), alfa smooth muscle actin (1:500, Abcam #ab7817), CD41 (1:1000; Abcam #ab11024) and fibrinogen (1:250; Abcam #ab118488). Antibodies were diluted in blocking buffer (5% goat serum/3% BSA in PBS), and slides were incubated at 4 °C overnight. Negative controls were made omitting the primary antibody. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa 488 and goat anti-mouse IgG conjugated to Alexa 594 (Life Technologies), used at 1:500. Each immunostaining procedure was performed in triplicate and repeated at least twice per vein sample.
The stained sections were mounted with prolong gold antifade (Invitrogen), containing DAPI for nuclear staining. Imaging was done using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera.