Wide field epifluorescence microscope

6-μm frozen sections of the harvested arteries were taken throughout the zone of injury. Frozen sections were fixed in acetone or 4% paraformaldehyde for 10 minutes before staining. Sections were then permeabilized with 0.2% Triton-X (Sigma-Aldrich). The sections were blocked in 10% goat serum. Blocking of endogenous avidin/ biotin was performed with a commercial kit (Vector, Burlingame, Calif) before incubation with one of the following primary antibodies: Ki67 (1:500; Abcam AB16667) and CD45 (1:150; Abcam AV10558). This was followed by incubation with a biotinylated goat-anti-rabbit secondary antibody (1:200; BioLegends, San Diego, CA). Conjugated streptavidin was applied to fluorescently label the secondary antibody (1:200; Dako). Fluorescently labeled sections were then mounted with DAPI mounting solution (SouthernBiotech).
Images of six sections per artery were obtained at 20x with a Nikon Widefield Epifluorescence microscope. Images were analyzed via ImageJ software. The intima/media was selected freehand, and the nuclei and antibody channels were separated. Areas of overlap between nuclei and antibody staining were designated as positive cells. The number of positive cells was normalized to total intima/media area.

Images were acquired using a Nikon wide-field epifluorescence microscope, Nikon A1R confocal, and Yokogawa W1 spinning disk confocal. All microscopes were run using Nikon Elements software. The widefield microscope was an inverted Nikon Eclipse TiE system with a 120BOOST LED-based illumination system and equipped with a Photometrics HQ2 CoolSnap camera and motorized XY stage. The Nikon A1R point scanning confocal system was run on an inverted Nikon Eclipse TiE base with 405-, 488-, 568- and 647nm excitation laser lines and four detectors: two GaAsP and two Alkali PMTs with a motorized XY stage. The Yokogawa W1 spinning disk confocal has an inverted Nikon Eclipse TiE base and 100mW 405-, 490-, 561-, and 640nm lasers, equipped with an Andor iXon 888 Life EMCCD camera linked with a 10-position filter wheel and a motorized XY stage. The spinning disk system was enclosed in an environmental chamber with temperature and local [CO2] control.