To monitor the cellular distribution of cyclin-D isoforms, pancreatic sections were stained with either an anti-cyclin-D3 antibody plus HRP–goat anti-mouse IgG and Alexa Fluor 488 tyramide (Life Technologies, Eugene, OR, USA), or an anti-cyclin-D1 antibody plus HRP–goat anti-rabbit IgG and Alexa Fluor 488 tyramide. The tyramide amplification steps were used to enhance the fluorescence signal and were performed according to the manufacturer’s instructions (Life Technologies). Pancreatic sections were co-stained with an anti-glucagon antibody raised in either mouse or rabbit (each from Abcam) and with guinea pig anti-insulin (Dako, Ely, UK) plus relevant secondary antibodies labelled with Alexa Fluor 568 and Alexa Fluor 647 (Life Technologies), respectively. Images were captured under fluorescence illumination using a Leica AF6000 microscope (Leica, Milton Keynes, UK).