Rabbit anti somatostatin

In all recordings, cell identity (α, β or δ) was subsequently established by immunocytochemistry. Biocytin (0.5 mg ml⁻¹) was included in the intracellular solution to allow identification of the cell recorded from. Following voltage-clamp experiments, islets were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) overnight and permeabilized with 0.3% Triton X-100. Non-specific binding was blocked by pre-treatment for 2 h with 5% normal goat serum before incubating with the different primary antibodies for 4–12 h (guinea pig anti-insulin (Abcam, Cambridge, UK), sheep anti-glucagon (Sigma-Aldrich, St Louis, MO) and rabbit anti-somatostatin (Vector Labs, Burlingame, CA)). After washing with PBS, the islet was incubated for 1 h in secondary antibodies (Alexa 633 goat anti-guinea pig (insulin), Alexa 405 goat anti-mouse (glucagon) and Alexa 543 goat anti-rabbit (somatostatin)). Biocytin labelling was visualized by using Alexa Fluor 488 conjugated streptavidin (0.04 mg ml⁻¹; Thermo Fisher). Islets were then washed and imaged on a confocal microscope (Axioskop 2 upright microscope fitted with a Zeiss LSM 510 meta confocal and a chameleon multiphoton module).

Human islets were fixed in 10% neutral‐buffered formalin, dehydrated, and processed for paraffin wax embedding and sectioning (3 μm). GLP‐1R immunoreactivity was detected using rabbit anti‐GLP‐1R (LS‐A‐1205 and LS‐A‐1206, Lifespan, 1:100 and 1:750, respectively) antibodies. Cell identity (α, β, or δ) was established by costaining for hormones. Islets were permeabilized with 0.3% Triton X‐100 and then incubated at 4°C with primary antibodies for 4–12 h (guinea pig anti‐insulin (Abcam, Cambridge, UK), sheep anti‐glucagon (Sigma‐Aldrich, St Louis, MO) and rabbit anti‐somatostatin (Vector Labs, Burlingame, CA). After washing with PBS, the islets were incubated for 1 h in secondary antibodies (Alexa 633 goat anti‐guinea pig (insulin), Alexa 405 goat anti‐mouse (glucagon) and Alexa 543 goat anti‐rabbit (somatostatin)). Islets were imaged on a confocal microscope (Axioskop 2 upright microscope fitted with a Zeiss LSM 510 meta confocal and a chameleon multiphoton module). Quantifying the colocalisation of GLP‐1R with insulin, glucagon and, SST was conducted manually by counting the number of coexpressing cells.