Human Patient SamplesFrozen sections of human pancreata were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative T1D research project sponsored by the Juvenile Diabetes Research Foundation International (JDRF). We used sample/CaseID as indicated on the nPOD website. A total of four T1D and five T2D samples were selected for this study based on their blood glucose, body mass index (BMI) values, and history of diabetes. Samples from healthy patients (n=5) were used as a control. All patient samples used in the study were excised from the tail of the pancreas.Immunohistochemistry of Pancreatic Sections and CellsHuman and mouse pancreatic sections were co-immunostained with IC2, anti-insulin and anti-glucagon or anti-somatostatin antibodies. Frozen sections (5–7-µm thick) were fixed with 4% formaldehyde for 5 min, washed with PBS and blocked with 2% BSA in PBS for 1 hr at room temperature. IC2 (1 µg/ml) and guinea pig anti-insulin (Abcam, Cambridge, MA) and rabbit anti-glucagon antibodies (Abcam), diluted to 1:50 and 1:100, respectively, were mixed in 2% BSA, added to the sections and incubated at room temperature for 2 hr. Then, sections were washed with PBS and incubated in the mixture of goat anti-rat IgM-AF594 (1: 1000 dilution, Invitrogen, Carlsbad, CA), goat anti-guinea pig IgG (H+L)-FITC (1:200 dilution, Abcam) and goat anti-rabbit IgG (H+L) AF-680 (1:500 dilution, Invitrogen) for 2 hr. Next, the sections were washed with PBS, mounted in ProLong® Gold anti-fade reagent with DAPI (Invitrogen), and observed under fluorescence microscope. For pancreatic sections from human patients, the fluorescence intensity was scored on a scale of 1 to 5 by two blinded investigators. In all of these experiments, an irrelevant purified rat monoclonal IgM was used as control for IC2. Staining without primary antibodies was also used as a control.