Phenotype of neointimal cells. (A) 2-color immunohistologic analysis of carotid artery sections from WT arteries taken on day 28 after injury. Consecutive sections were stained with anti–α-SMA (red) and either anti-VWF, anti-CD31, anti–P-selectin, or anti–E-selectin (green). Yellow indicates colocalization. Original images taken at ×100 magnification. (B) 3-color immunohistologic analysis of single cells isolated from intima of control uninjured (top row) and day 28–injured arteries (bottom row) from WT mice. Cells were prepared after first scraping off and discarding the luminal layer. All sections were stained with DAPI (blue), anti–α-SMA (red), and (green) either anti-VWF, anti–P-selectin, or anti–E-selectin. Yellow indicates colocalization. Original images taken at ×400 magnification and then enlarged 5× digitally. As indicated by the white lines, images of individual cells have been cut and spliced into a single frame in Adobe Photoshop (San Jose, CA). (C) 2-color immunohistologic analysis of carotid artery sections from WT arteries taken on day 28 after injury. Dual exposure image is on the right. Consecutive sections were stained with anti–α-SMA (red) and (green) either anti-CD41 (top row) or anti-CD34 (bottom row). Yellow indicates colocalization. Original images taken at ×100 magnification. (D) 3-color immunohistologic analysis of carotid artery sections from WT arteries taken on day 21 after injury. Injured mice were injected with 7.5 × 105 CD34+ cells from α-TFPI–Tg mice on day 7 (top row) or day 14 (bottom row). All sections were stained with DAPI (blue), anti–α-SMA (red) and anti-TFPI (green). (A-D) Representative of experiments performed on at least 2 independent cohorts of animals.
