In order to evaluate NRB-AF12 co-localization with endoplasmic reticulum, Golgi apparatus and with the sulfonylurea receptor (SUR) subunits of the ATP-sensitive potassium channels, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with ice cold methanol for 5 min at −20°C. ER was highlighted with antibodies directed against the ER resident protein calreticulin (1:200; Abcam #ab2907); Golgi apparatus was highlighted with antibodies directed against the Golgi apparatus structural protein GM130 (1:500; BD Biomedsciences #610822); SUR2 subunits of KATP channel were highlighted using either anti-SUR2A (1:750; Abcam #ab174629) or anti-SUR2B (1:750; Abcam #ab174631) antibodies. All primary antibodies were incubated for 90 min at room temperature. Following a PBS wash, cells were incubated with Cy™3 goat anti-rabbit IgG fluorescent secondary antibody (1:500; Jackson ImmunoResearch #111-165-003) or with Cy™3 goat anti mouse IgG fluorescent secondary antibody (1:500; KPL #072-01-18-06) for 60 min at room temperature in the presence of either 500 ηM NRB-AF12 or 500 ηM ER-Tr green. Finally, coverslips were mounted on microscope slides using Mowiol 40–88 (Sigma Aldrich). Confocal images were acquired with a Laser Scanning Confocal Microscope (LSCM) D-Eclipse C1 SHV Nikon system equipped with a Nikon Eclipse E600 microscope and 3 laser diode modules (488/543/637 nm), using a CFI Plan Apochromat 60x NA 1.4 objective and analyzed using NIS Elements software (Nikon) and the open-source platform for biological-image analysis Fiji software.