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Immunofluorescence and confocal microscopy Cells were labeled at 37 °C with 1 μM SNAP-Surface 488 in HEPES-bicarbonate buffer (120 mM NaCl, 4.8 mM KCl, 24 mM NaHCO 3 , 0.5 mM Na 2 HPO 4 , 5 mM HEPES, 2.5 mM CaCl 2 , and 1.2 mM MgCl 2 , saturated with 95% O 2 /5% CO 2 , pH 7.4) plus 3 mM glucose and 1% BSA, stimulated with agonists at 11 mM glucose for the indicated times, fixed and processed for immunofluorescence with mouse anti-Rab11 (610657, BD Biosciences, 1/20) or anti-rodent GLP-1R monoclonal antibody (Mab 7F38, DSHB, 2 μg μl −1 ) plus Alexa Fluor 546 secondary (Life Technologies, 1/200), mounted in Prolong Diamond antifade reagent with 4,6-diamidino-2-phenylindole (Life Technologies) and imaged with a Zeiss LSM-780 inverted confocal laser-scanning microscope in a ×63/1.4 numerical aperture oil-immersion objective and analyzed in Image J