Anti somatostatin

Pancreas tissue was fixed in buffered 10% formalin, processed to wax blocks and sectioned onto slides (4 μm). Dual fluorescence staining was performed to determine the islet cell type in which β-galactosidase/GPR120 was expressed. Anti-insulin (1:500; Dako, Cambridge, UK), anti-glucagon (1:200; Abcam, Cambridge, UK) and anti-somatostatin (1:100; Millipore, Watford, UK) were detected using Alexa Fluor-488 conjugated anti-goat antibody (Abcam) and anti-β-galactosidase (1:1200; Abcam) with Alexa Fluor-568 conjugated anti-rabbit antibody. Citrate buffer (pH 6) was used for antigen retrieval in the case of β-galactosidase and somatostatin. Briefly, tissue sections were fixed, de-waxed and rehydrated prior to antigen retrieval (heating in a microwave at full power for 20 min followed by cooling to room temperature). The sections were blocked before the addition of primary antibodies and washed prior to the addition of secondary antibodies and DAPI nuclear stain (1:500; Thermo Scientific). Slides were mounted in Hardset Vectashield Mounting medium (Vector Laboratories, Peterborough, UK) and analysed on a Nikon Eclipse 90i Fluorescent Microscope.

For immunocytochemistry, single cells were fixed in 2.5% paraformaldehyde and kept at 4 °C. On the day of the experiment, cells were permeabilised with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min at room temperature. Permeabilisation was followed by blocking of nonspecific binding with PBS containing 5% swine serum for 20 min. The cells were then incubated overnight with anti-SGLT2 antibodies (1:75, Santa Cruz Sc-393350) at 4 °C. On day 2, cells were incubated with mouse Alexa-488 Tyramide kit (T20948, Molecular Probes) according to manufacturer’s instruction. Cells were then incubated in primary antibodies anti-somatostatin (1:100, Millipore, MAB354) for 1 h at room temperature, followed by secondary antibodies Alexa 568 anti-rat (1:500, A-11077, Invitrogen) for 30 min by the nuclear stain RedDot2 (1:200, 40061, Biotium) for 10 min at room temperature. Samples were visualised with a Bio-Rad confocal microscope using appropriate lasers and filters.

Animals (n = 2 in each condition) were perfused with phosphate-buffered saline (PBS, pH 7.4) containing 4% (w/v) paraformaldehyde (PFA) and 0.2% (v/v) picric acid under general ketamine-medetomidine anesthesia. The brain was removed, incubated for 24 h in perfusion solution, and infiltrated with 30% (w/v) sucrose in PBS for at least 24 h. Coronal sections of 50 µm were cut on a Leica SM 2000R sliding microtome. The sections were rinsed three times in Tris-buffered saline (TBS, Sigma), three times in TBS containing 3% (w/v) Triton X-100 (TBS-T), and once for 1 h in TBS-T containing 20% (v/v) horse serum (Vector Labs) and then incubated for 48 h at 4°C in TBS-T containing 1% horse serum and combinations of the following primary antibodies: anti-GFP (chicken, 1∶500, AbCam), anti-parvalbumin (mouse, 1∶2,000, Swant), or anti-somatostatin (rabbit, 1∶500, Millipore). The sections were rinsed 4 times in TBS and stained in TBS-T containing 1% horse serum and Alexa488- and Alexa546-labeled secondary antibodies (Invitrogen). After four rinses in TBS, the slices were mounted in VectaShield (Vector Labs) and imaged on a Leica TCS SP5 confocal microscope.