Immunostained sections were examined with a Nikon Eclipse E600 epifluorescence-bright field microscope and digital images recorded. Group assignment was blinded to the investigator, except groups 1 and 4 where many islets were insulin-deficient. From each pancreatic section, islets with approximately ≥20 endocrine cells were imaged for the presence of IL-1β, insulin, glucagon, CD68 cells (macrophage marker) and CD3 cells (T cell marker). Each of the specific image sets from multiple acquisitions were merged with Adobe Photoshop CS6 (Adobe Systems, San Jose, CA, USA) following conversion of IL-1β immunostained cells to a greyscale fluorescence mode. Fifteen exocrine fields that were devoid of islet and ductal areas were selected randomly by microscopically traversing different regions of each pancreatic section and imaged at ×20 objective. The number of IL-1β-positive cells in each field was enumerated and the mean number per field per section determined. All data were included in the study. For each section, the number of IL-1β-positive cells in the peri- and intra-islet regions was enumerated and the mean number of cells per insulin-positive and -negative islet determined. Islets that showed immunostaining for IL-1β in alpha cells were scored microscopically as negative, weak or moderate intensity.

The immunohistochemical specificity of anti-IL-1β (catalogue no. 12242; Cell Signaling Technology) has been validated by the manufacturer. Thus, by immunohistochemistry, it successfully detects IL-1β immunoreactive cells in human colonic sections from individuals with chronic colitis. Further, by western blotting, the same antibody detects mature recombinant human and mouse IL-1β and the human precursor (molecular mass of 34 kDa) in extracts of Raw 264.7 and human monocyte-derived THP-1 cells, following exposure to Brefeldin and lipopolysaccharide (LPS), respectively. Anti-IL-1β was replaced with diluent to act as negative controls. As further controls, paraffin sections of THP-1 cells, harvested after incubation with LPS (100 ng/ml for 3 h) and without (from Cell Signaling Technology), were evaluated in this laboratory for IL-1β immunohistochemical specificity. The immunostaining protocol for IL-1β was tested with formalin-fixed tonsil sections, supplied by Department of Surgery, University of Auckland. Sections from selected pancreas and tonsil were immunostained for IL-1β in two separate sections. They were then exposed to rabbit anti-CD68 or rabbit anti-CD3, followed by donkey anti-rabbit IgG-biotin and then streptavidin–Alexa 568. Pancreatic sections were immunostained subsequently for insulin as above, followed by donkey anti-guinea pig IgG–Alexa 488.