Immunofluorescence Imaging of HUVEC Cells

Typically, ∼5 × 10⁴ HUVEC were plated on 0.2% gelatin-coated 4-well chamber slides (Nunc, Thermo Scientific) and grown to full confluence in their growth media at 37 °C. Cells were treated as per the experimental conditions contained herein, and immunofluorescence was performed as previously done (100 (link), 101 (link)). Slides were incubated with conjugated secondary antibodies such as: goat anti-rabbit IgG Alexa Fluor® 488 and goat anti-mouse IgG Alexa Fluor® 564 (Invitrogen). Nuclei were visualized with 4′,6-diamidino-2-phenylindole (Vector Laboratories). JC-1 live cell immunofluorescence images were acquired with a ×10 objective on a LEICA DM5500B microscope installed with the Leica Application suite, using advanced fluorescence version 1.8 software (Leica Microsystems, Frankfurt, Germany). Confocal analyses were carried out utilizing a ×63, 1.3 oil-immersion objective of a Zeiss LSM-780 confocal laser-scanning microscope with Zen Imaging Software. A full description of the immunofluorescence and confocal laser microscopy protocol can be found elsewhere (78 (link)).