Anti insulin antibody

After fixation, pancreas samples were embedded in paraffin and were stained with hematoxylin and eosin (H&E). Immunohistochemical staining of α-cells, β-cells, and CCAAT/enhancer-binding protein homologous protein (CHOP) were performed in the Kotobiken Medical Laboratories, Inc. (Tokyo, Japan) using anti-glucagon antibody (Takara Bio Inc., Shiga, Japan), anti-insulin antibody (Takara Bio Inc.), and anti-CHOP antibody (Proteintech Group Inc., Chicago, IL). Specimens were examined using a microscope (200× magnification). Images were captured using an Olympus DP21 camera system (Olympus, Tokyo, Japan), and immunoreactivities were quantified using image analysis software (Image J, United States National Institutes of Health). The mean area (µm 2 ) of the pancreatic islets and their distribution were determined in all H&E-stained sections. The percentages of α-and β-cell areas were calculated as follows: {glucagonor insulin-positive area (µm 2 )/islet area (µm 2 )} × 100. The glucagon-and insulin-positive areas were quantitated using an appropriate threshold after altering and adjusting color images to gray scale.