All pancreas samples were fixed and cryo-protected in 30% sucrose overnight before freezing, as described before [20 (link), 33 (link), 34 (link)]. Staining with 3,3′-Diaminobenzidine (DAB) was performed using a DAB chromogen system (Dako, Carpinteria, CA, USA). A Biotin-TSA or Cy5-TSA enhancer kit (Fisher Scientific) was used to amplify VEGF signals. GFP was detected by direct fluorescence. Primary antibodies for immunostaining were: guinea pig polyclonal anti-insulin and anti-pancreatic polypeptide (Dako), goat polyclonal anti-VEGF-A (R&D systems, Minneapolis, MN, USA) and anti-insulin (Santa Cruz, CA, USA); rabbit polyclonal anti-somatostatin (Dako) and anti-VEGF-A (Santa Cruz); rat polyclonal anti-CD31 (BD, San Jose, CA, USA) and mouse monoclonal anti-glucagon (Sigma, St Louis, MO, USA). Secondary antibodies were all purchased from Jackson ImmunoResearch. Nuclear staining was performed with Hoechst (BD). When duct cells were labelled with biotin-dolichos biflorus agglutinin (DBA) (Vector Lab, Burlingame, CA, USA), they were detected with Cy5-conjugated streptavidin. Staining, imaging of sections and quantifications were performed as described previously [20 (link), 33 (link), 34 (link)].