2.6. Immunofluorescence

Dispersed cells were fixed for 1 h at room temperature with 4% paraformaldehyde and then permeabilized for 45 min with Triton X-100 (0.025%). Nonspecific binding was blocked with normal goat serum (NGS 10%) before immunostaining. Dispersed cells were immunolabeled overnight at 4 °C for glucagon, GCaMP6f, and SST with a mouse monoclonal anti-glucagon antibody (1:2500, Merck G2654), a rabbit polyclonal anti-GFP antibody (1:2000, Novus Biologicals NB600-308) and a rabbit polyclonal anti-SST antibody (1:400, Abcam ab108456), respectively. After washing the primary antibodies, the cells were treated for 2 h at room temperature with secondary antibodies. Glucagon was revealed in red using Alexa Red 594-conjugated goat anti-mouse IgG (1:500, Thermo Fisher A11020), whereas GCaMP6f and SST were revealed in green using Alexa Green 488-conjugated goat anti-rabbit IgG (1:100, Thermo Fisher A11070).