2.3. Confocal imaging

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.