FIGURE 3.

Immunofluorescence-based quantification revealed regulated PMCA4 splicing in VSMC of injured carotid arteries. A, confocal images of uninjured and injured carotid artery cross-sections from male C57BL6/J mice stained for PMCA4a/e, PMCA4b, smooth muscle marker (sm22α), proliferation marker (proliferating cell nuclear antigen), and collagen deposition (trichrome). Scale bars in panel A trichrome images = 500 μm, in confocal images = 20 μm, and in panel D confocal images = 50 μm. B, confocal microscopy Z-stack quantification of PMCA4a/e staining. Each bar represents a mean of 3 mouse carotid artery cross-sections in which ∼40–70 cells or cell clusters were used to quantify fluorescent intensity through 20–40 focal planes of 5-μm-thick sections. C, morphometric analysis of injured carotid arteries in PMCA4 wild-type (P4WT) and PMCA4 knock-out (P4KO) mice (n = 4). Carotid artery cross-sections were immunostained for PMCA4a/e with media and adventitia captured as regions of interest for area measurements on FluoView microscope (40× objective) and software (Olympus, Mountain View, CA). The ratio of adventitia:media areas are decreased in injured carotid arteries of P4KO mice (P4WT = 3.1 ± 0.5 versus PMKO = 1.4 ± 0.1; p = 0.01; n = 4). D, representative immunofluorescent micrographs of P4WT and P4KO injured carotid arteries immunostained for PMCA4a/e show reduced medial thickening in P4KO mice.