Immunohistochemistry and image digitalisationEach pancreas received was divided into a head, body and tail region, each of which was subjected to serial transverse sectioning. Within each region, tissue pieces were consecutively and alternately used for preparation of both formalin-fixed paraffin-embedded and frozen tissue blocks. Three consecutive paraffin sections were cut at 4 µm from one representative formalin-fixed paraffin-embedded tissue block within each region. All sections were deparaffinised and rehydrated with serial passage through changes of xylene and graded ethanol. All slides were subjected to heat-induced antigen retrieval in Target Retrieval Solution (Dako, Carpinteria, CA, USA). The tissue sections were double stained for insulin (polyclonal guinea pig anti-insulin,1:2000 dilution; catalogue no. A0564, RRID:AB_10013624; Dako) and one of the following markers: CD68 for macrophages (monoclonal mouse anti-CD68, 1:2000 dilution; catalogue no. M0814, RRID:AB_2314148; Dako); CD45 for leucocytes (monoclonal mouse anti-CD45, 1:200 dilution; catalogue no. M0701, RRID:AB_2314143; Dako) or Ki67 for DNA replication (monoclonal mouse anti-Ki67, 1:160 dilution; catalogue no. M7240, RRID:AB_2142367; Dako) as previously described [8]. Antigen–antibody binding was visualised using the EnVision G/2 Doublestain (peroxidase-DAB and alkaline phosphatase-Permanent Red; catalogue no. K5355; Dako) polymer system. Subsequently, the slides were counterstained with Mayer’s Hematoxylin (catalogue no. S3309; Dako), dehydrated in ethanol and mounted with Cytoseal XYL media (Richard-Allan Scientific, Kalamazoo, MI, USA). Stained slides were then digitalised and processed in preparation for statistical analysis (see ESM Methods for image acquisition and processing details).