Isolation and Characterization of Pancreatic Islets

Pancreatic islets were isolated from 8 to 10-week-old male C57BL/6 mice as previously described (Jun et al., 1999 (link)). Intact islets were dissociated at 37°C in Accutase (Millipore), given STZ (1 mM) for 15 h, washed with fresh media, and then cultured with BTC (1 nM). The islet cells or the αTC1-9 cells were fixed in 4% paraformaldehyde, permeabilized in permeabilization buffer (Thermo Fisher Scientific), blocked in blocking solution (Thermo Fisher Scientific), and then incubated with mouse anti-glucagon (Sigma, 1:100), rabbit anti-glucagon (DAKO, 1:100), mouse anti-PDX-1 (DSHB, Iowa, IA, 1:100) or mouse anti-BrdU (DAKO, 1:50) antibodies. FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, 1:200) or TR-conjugated goat anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology, 1:200) were used as secondary antibodies. Fluorescence was imaged using a laser scanning confocal fluorescent microscope (LSM 700).