Rabbit monoclonal anti glucagon

Liver and pancreas were taken, fixed with 10% formalin, and paraffin-embedded sections were prepared for further analysis. Paraffin sections were stained with H&E and Periodic Acid-Schiff (PAS) for morphology analyses. For the immunohistochemical staining, the following primary antibodies were used: guinea pig polyclonal anti-insulin (Abcam) and rabbit monoclonal anti-glucagon (Abcam). The following secondary antibodies were used: Alexa Fluor 488–conjugated AffiniPure Goat Anti-Guinea Pig (Jackson Immunoresearch) and Alexa Fluor 594–conjugated AffiniPure Goat Anti-rabbit IgG (Jackson Immunoresearch). The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI), and sections were preserved using fluorescence mounting medium (Electron Microscopy Science). Images were acquired on a Nikon 90i Upright. ImageJ was used to process the images.

The islet structure in the pancreas was identified by confocal imaging and immunohistochemistry. The experiments were carried out following the recently published protocol [17 ]. Briefly, the entire pancreas was isolated, paraffin-embedded, cut into 5 μm sections and rehydrated for staining with primary antibodies of guinea pig anti-insulin (Eurodiagnostica), rabbit monoclonal anti-glucagon (Abcam). The secondary antibodies, donkey anti-guinea pig with conjugated cy2 (Jackson Immuno-lab), donkey anti-rabbit with conjugated cy5 (Jackson Immuno-lab) and Hoechst 33258 (Life Technologies), were used to detect insulin, glucagon, and nuclei, respectively. The images were acquired by confocal microscope (Meta 510, Zeiss, Germany) and proceeded by ZEN 2012 software.