Afv10i confocal microscope

MM.1S and NCI-H929 cells were fixed in PBS with 4% paraformaldheyde and permeabilized in 0.1% Triton X-100. After blocking with PBS containing 5% BSA, cells were incubated overnight at 4 °C with the following primary antibodies: anti-MAPK4 (Abcam) followed by the incubation with the proper secondary antibody fluorescein isothiocyanate (FITC)-conjugated for 30 min. Cells were then washed with PBS and nuclei stained by DAPI (Sigma). Cells were observed and photographed under aFV10i Confocal microscope (Olympus, Japan).

The CFs cocultured with EPC-Exos were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% BSA, the cells were incubated with the following primary antibodies: anti-α-smooth muscle actin (SMA, Abcam, ab5831), anti-vimentin (Abcam, ab92547), anti-CD31 (Dako, M0823), anti-vascular endothelial growth factor receptor 2 (VEGFR-2, Abcam, ab10972), anti-p53 (Abcam, ab131442), or anti-JMY (Abcam, ab217953) overnight at 4°C. The cells were incubated for 30 mins with the appropriate secondary antibody conjugated to fluorescein isothiocyanate (FITC). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Briefly, the cells were washed twice with PBS and incubated for 5 min in the dark with 1 : 750 solution of stock DAPI (2 mg/mL). After rinsing with PBS, the slides were allowed to air dry at room temperature, and the cells were observed under an Afv10i confocal microscope (Olympus, Japan).