Islet β-cell proliferation

Groups of 250 mouse or human islets were incubated for 48 h at 37 °C (95% air/5% CO₂) in RPMI-1640 with 2% FBS (mouse) or CMRL with 0.2% albumin (human), supplemented with 10 μM SR141716A, 10 μM AM251 or vehicle (0.0001% DMSO). Islets were then pelleted at 135 g, fixed with 4% paraformaldehyde and embedded in paraffin. Sections of 5 μm thickness were dewaxed, then antigens were retrieved using citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0). Sections were incubated overnight at 4 °C with primary anti-insulin (guinea pig) and anti-Ki67 (rabbit) antibodies at 1:200 dilution, then incubated with anti-guinea pig AlexaFluor 594 and anti-rabbit AlexaFluor 488 antibodies (1:150 dilution) for 1 h at room temperature. The primary and secondary antibodies are listed in Suppl. Table S3. Images were visualized using a Nikon A1 Inverted Confocal microscope and analysed blindly before quantification using Fiji Image J software (https://fiji.sc) [4]. For each experiment, the images were acquired with the same settings and histological quantifications were performed in paraffin sections that had been immunostained under the same conditions.