Pancreatic tissue was fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL) and embedded in paraffin for sectioning and processing as previously described (Albury et al. 2015 (link)). Slides were stained for hematoxylin and eosin (Surgipath) or immunohistochemistry using the Polymer Refine Detection reagents (Leica) on the Bond-Max immunostainer (Leica). Antigen retrieval was optimized using sodium citrate (pH 6) or EDTA (pH 9) buffers (Leica). The following antibodies were used: phospho-Akt Ser473 (GeneTex, Irvine, CA), also phospho-mTor Ser2448, phospho-S6 Ser235/236, glucagon, and insulin (all from Cell Signaling Technology, Danvers, MA). All slides processed on the immunostainer were run with a negative control, which was treated with antibody diluent instead of the primary antibody, to ensure antibody specificity. Images were taken using a Leica DM 2000 microscope with 5X, 10X, or 40X objectives. Islet size was measured in the whole pancreas of three mice per genotype. The mice selected had no significant lesions (NSL) at the time of necropsy, as described by a pathologist. The pancreas was sectioned into 5μm sections and every 25th section was H&E stained. A total of 50 islets from three sections were analyzed per mouse. Islet diameter was determined using measuring tools available on an Axio Imaging System (Zeiss, Oberkochen, Germany).