Pancreas samples were fixed in formaldehyde and embedded in paraffin for subsequent analysis. Five-micrometer sections were stained with hematoxylin and eosin or by immunohistochemistry for islet hormones using a cocktail of antibodies to insulin, glucagon, or somatostatin (21 (link)). In addition, sections were immunostained for insulin, Ki67, or DAPI (nucleus) to assess proliferation, TUNEL for apoptosis, and for duct marker using anti-CK19 antibodies, and GLP-1 to identify incretin immunoreactivity. The hematoxylin and eosin slides were examined in all cases by two pathologists to exclude those with pancreatitis, autolysis, and tumor infiltration.
Primary antibodies included insulin (guinea pig antibody, 1:200; Abcam), glucagon (mouse mono, 1:500; Sigma-Aldrich), somatostatin (rabbit poly, 1:500; Abcam), or Ki67 (mouse mono antibody, 1:50; BD Biosciences), GLP-1 (rabbit antibody, 1:1000; J.F. Habener, MD, Massachusetts General Hospital, Boston, MA), and CK19 (rabbit poly, 1:100; Abcam). For TUNEL we used an Apoptag Fluorescein in situ apoptosis detection kit (Roche) and PDX1 (rabbit poly, 1:200; Cell Signaling).
Secondary antibodies were donkey anti–GP-594, donkey anti–mouse-AMCA, donkey anti–rabbit-488, donkey anti–mouse-AMCA, and biotinylated donkey anti-rabbit (all from Jackson ImmunoResearch, West Grove, PA), and peroxidase labeled polymer (Dako).
Mezza T., Muscogiuri G., Sorice G.P., Clemente G., Hu J., Pontecorvi A., Holst J.J., Giaccari A, & Kulkarni R.N. (2014). Insulin Resistance Alters Islet Morphology in Nondiabetic Humans. Diabetes, 63(3), 994-1007.
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