A1 confocal microscopy system

Enzymatically-isolated coronary arterial myocytes were plated on glass microscope slides and allowed to adhere for 20 min at room temperature before fixation with 4% paraformaldehyde in phosphate buffered saline (PBS). Following fixation, cells were incubated in PBS containing Alexa 555-conjugated wheat germ agglutinin (WGA; 5 μM) for 10 min at room temperature and washed twice with PBS for 5 min. Non-permeabilized cells were then blocked with 1.5% BSA in PBS for 45 min. Immunofluorescent labeling of freshly isolated arterial myocytes was performed as described previously [19 (link)] using a rabbit polyclonal antibody specific for an extracellular epitope of K_V1.5 (Alomone; APC-150). The secondary antibody was an Alexa Fluor 488-conjugated donkey anti-rabbit (Abcam; 5 mg/ml). Cells were imaged (512 × 512 pixel images) using a Nikon A1 confocal microscopy system coupled with a Nikon 60X oil immersion lens (NA = 1.4). Images were collected at multiple optical planes (z-axis step size = 0.5 μm). Specificity of secondary antibodies for anti-K_V1.5 primary antibodies was tested in negative control experiments in which the primary antibody was substituted with PBS. K_V1.5-associated fluorescence was not observed under these experimental conditions. Images were taken under identical laser power, PMT gain, and pinhole size for all cells analyzed.

To determine cellular uptake and subcellular localization of MSVs, cardiovascular cell lines were seeded into eight-well chamber culture slides (BD Biosciences, Franklin Lakes, NJ, USA) and incubated with a ratio of 25 MSVs per cell (3.8 μg/mL). Cells were collected at predetermined time-points, fixed, and stained. Images were obtained using a Nikon A1 confocal microscopy system (Nikon Instruments, Melville, NY, USA). Details provided in the Supplementary material online, Methods S1.