Mouse monoclonal anti glucagon

LUXendin555 (250 nM) or LUXendin645 (50–250 nM) were applied to cells or tissue for 60 min, before fixation in 4% formaldehyde. Primary antibodies were applied overnight at 4 °C in PBS + 0.1% Triton + 1% BSA. Secondary antibodies were applied in the same buffer for 1 h at room temperature, before mounting on slides using Vectashield Hardset containing DAPI. Primary antibodies were mouse monoclonal anti-GLP1R 1:30 (Iowa DHSB; mAb #7F38), rabbit anti-insulin 1:500 (Cell Signaling Technology, #3014), mouse monoclonal anti-glucagon 1:2000 (Sigma-Aldrich, #G2654), and mouse anti-somatostatin 1:5000 (Invitrogen, #14-9751-80). Secondary antibodies were goat anti-mouse DyLight488, goat anti-mouse Alexa Fluor 568, and donkey anti-rabbit DyLight 488 1:1000. Images were captured using an LSM880 meta-confocal microscope. DyLight488 and Alexa Fluor 568 were excited at λ = 488 nm and λ = 568 nm, respectively. Emitted signals were detected at λ = 500–550 nm (DyLight 488) and λ = 519–574 nm (Alexa Fluor 568). GLP1R surface expression was quantified vs. total GLP1R expression, and normalized against Exendin4(1–39) controls.

INS-1 cells (1 × 10⁶ cells/condition) were permeabilized and fixed with BD Cytofix/cytoperm buffer (BD biosciences, Cat No. 554714, San Diego, CA, United States) according to manufacturer’s instructions. Cells were incubated overnight at 4°C with the following primary antibodies diluted in cytoperm buffer: rabbit monoclonal anti-insulin 1:400 (Abcam, Cat No. ab181547, CA, United States) and mouse monoclonal anti-glucagon (Sigma-Aldrich, Cat No. G2654, Israel) 1:400 dilution in PBS. After washing, cells were incubated for 1 h at room temperature and protected from the light with the following secondary antibodies diluted 1:500 in cytoperm buffer: Alexa Fluor anti-rabbit AF-488 (Invitrogen, Cat. No. 21206, Eugene, OR, United States) and anti-mouse, AF-594 (Invitrogen, Cat. No. 11032, Eugene, OR, United States). Isotype controls were used to set-up negative gate: Rabbit IgG AF-488 1:400 dilution (Thermo Fischer Scientific, N°A21206, Carlsbad, CA, United States; and BD Horizon PE-CF594 Mouse IgG, k isotype control 1:400 dilution (BD bioscience, Cat No. 562292, San Diego, CA, United States). Cells were re-suspended in 500 μL FACS buffer (PBS+10%FBS). Flow cytometry data were acquired on LSR Fortessa X20 (BD Biosciences) and analyzed using Kaluza software.