Fluor save mounting medium

Proliferative β-cells were detected by immunostaining of transfected islet cells with rabbit anti-Ki67 (Abcam ab15580, 1:1500), guinea pig anti-insulin (Dako A0564, 1:100), goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher A-11008, 1:500), and goat anti-guinea pig IgG Alexa Fluor 555 (Thermo Fisher A-21435, 1:500) antibodies. Coverslips were mounted on microscope glass slides with Fluor-Save mounting medium (VWR International SA) and were visualized with a Zeiss Axiovision fluorescence microscope. Prolactin (500 ng/mL, Sigma) was used to stimulate proliferation for 48 h. A minimum of 800 cells were counted per condition.

MIN6B1 cells were seeded on poly-L-lysine-laminin-coated glass coverslips. BrdU (Roche) was added to the incubation medium for the last 6 h of culture. Thereafter, MIN6B1 cells were fixed in cold methanol and permeabilized using PBS supplemented with 0.5% saponin for 15 minutes. The coverslips were incubated in blocking buffer (PBS containing 0.5% saponin and 1% BSA) for 30 min and then sequentially exposed to a mouse anti-BrdU antibody (diluted 1/1400, Cell Signaling) for 1 h and to a goat anti-mouse Alexa Fluor 555 antibody (diluted 1/400, Invitrogen) for another 1 h. Finally, the cell nuclei were stained with Hoechst 33342 (1 μg/ml, Invitrogen) for 1 minute. Coverslips were mounted on microscope glass slides with Fluor-Save mounting medium (VWR International SA) and were visualized with a Zeiss Axiovision fluorescence microscope.