2.2. In situ hybridizationTo detect IL-1β mRNA expression level, RNAscope® 2.0 High Definition BROWN Assay (ACD, Hayward, CA) was used according to the manufacturer’s instructions (see Supplementary material for more details).

2.3. Indirect immunofluorescencePancreas sections were subjected to a standard triple indirect immunofluorescence (IF) staining protocol to determine the expression of IL-1β at the protein level and its localization in α and β cells (see Supplementary Materials for more details).

Immunofluorescence (IF) Image analysis was performed with Image Pro Premier software on randomly selected islets. For each islet, the difference of the mean intensity in the positive area (MIP), and the mean intensity in the negative area (MIN) (background) was measured (MIP-MIN). The differences between the groups and within the regions of each section were presented as the mean of (MIP-MIN) ± SD. All techniques, image acquisition and analysis have been validated by histology and microscopy specialists, at LJI which enabled us to rightfully quantify the IL-1β expression level.

2.4. AnalysisIn situ hybridization (ISH) Islets were quantified by Image-pro Premier software (Media Cybernetics, Rockville, MD). For each islet, the percentage of the positive area (or positive staining, defined by Image-pro Premier software) within the islet boundary was assessed and the differences between the groups and within the regions of each section were presented as the mean of analyzed islets (mean±SD).