Tissue harvest and immunohistochemical analysis

Tissues were harvested and fixed in paraformaldehyde (PFA) 4% overnight after transcardial perfusion with PBS for 2 min and ice-cold PFA 4% for 10 min. After fixation, tissues were washed in 70% ethanol and embedded in paraffin, for subsequent sectioning (5 µm thickness; Wax-It Histology Services, Vancouver, Canada). Brains were left in PBS containing 20% sucrose for 4 days and then frozen at −40 °C in 2-methylbutane prior to sectioning at 30 µm on a sliding microtome, in a one-in-six series through the rostrocaudal extent of the hypothalamus and immunostained by standard method⁵⁶. For immunofluorescence of paraffin-embedded tissues, sections were deparaffinized in xylene and rehydrated in graded ethanol before heat-induced epitope retrieval in an EZ Retriever microwave oven (BioGenes, Fremont, CA; 95 °C for 15 mins in 10 mM citrate buffer with 0.05% Tween-20). We blocked slides in DAKO Protein Block, Serum Free (Dako, Burlington, Canada) and incubated overnight in primary antibody diluted in DAKO Antibody Diluent followed by incubation in secondary antibody for 1 hour at room temperature. Slides were mounted and counterstained with nuclear stain 4′,6-diamidino-2-phenylindole (DAPI) and VECTASHIELD^® Hard Set Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA), and images were captured and analyzed with an ImageXpress^® Micro XLS System, controlled by MetaXpress^® High-Content Image Acquisition & Analysis Software (Molecular Devices Corporation, Sunnyvale, CA) with a scientific CMOS camera, a Nikon 20 × Plan Apo objective (NA = 0.75, 1-6300-0196; Nikon, Tokyo, Japan), and DAPI (DAPI-5060B), FITC (FITC-3540B), Cy3 (Cy3-4040B), Texas Red (TXRED-4040B), and Cy5 (Cy5-4040A) filter cubes. We performed quantification of histological images in the same software (MetaXpress^®). We used an automated journal (multiwavelength cell scoring) that identifies all cells by DAPI fluorescence, then we defined parameters for cell diameter, total area, and intensity of IAPP or insulin immunoreactivity to identify IAPP + or insulin+ cells and IAPP + or insulin+ area. Finally we assessed the presence of fluorescent protein immunoreactivity in insulin+ or insulin- cells to assess recombination rate (Figs. 3a,d, and ​and4d),4d), assessed the intensity of insulin immunoreactivity in IAPP + cells (Fig. 6h), and/or calculated the % IAPP + area relative to the total pancreas area using autofluorescence to detect the total pancreas area (Fig. 6h). All antibodies used are listed in Table ^S1.