Dapi mounting solution

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.

Cells are washed with 1x Phosphate-buffered saline (PBS) and fixed with cold 1:1 acetone (#A929-1, Thermo Fisher Scientific)/methanol (#A412P-4, Thermo Fisher Scientific) for 20 min at −20°C. Cells are then blocked in 5% bovine serum albumin (BSA, #BP9706100, Thermo Fisher Scientific) overnight at 4°C. Before staining, the cells are scanned through Celigo S Imaging Cytometer in order to verify the absence of any immunofluorescence signal. E-cadherin (#3195S, Cell Signaling Technology) and Trop2 antibodies (sc-376746, Santa Cruz) are added in 1:200 dilution in PBS onto the cells for overnight incubation at 4°C. Cells are further washed three times for 5 min with 1xPBS and secondary antibodies anti-rabbit Alexa flour 594 (#NC0208612, Fisher Scientific) and anti-mouse Alexa flour 594 are applied for 2 hours at room temperature. After three times washing with 1xPBS for 5 min each, cells are mounted with 20% DAPI mounting solution (#0100-20, SouthernBiotech) and 20% glycerol (#BP229-1, Thermo Fisher Scientific) in 1xPBS.