Anti glucagon

Pancreatic sections were incubated with 5% BSA for 1h at room temperature and then incubated overnight with the anti-insulin (Proteintech, 15848-1-AP), and anti-Glucagon (Proteintech, 15954-1-AP) at 4°C. The next day, sections were stained with the secondary antibody for 1 hour at room temperature in the dark. The nucleus was stained with 4, 6 diamidino-2-phenylindole (DAPI) at room temperature for 5 minutes. The tissue sections and cells were imaged under a fluorescence microscope (BX61, Olympus, Japan).

All samples were imaged using an Olympus BX41 microscope. Image processing, quantification of pancreatic islet areas, and oil red staining were performed using ImageJ (39 ).
For immunofluorescence, deparaffinized and rehydrated sections were subjected to a citrate-based antigen retrieval. After permeabilization and blocking, sections were incubated with primary antibodies (anti-insulin (Cat #66198-1-Ig, Proteintech Group, Inc); anti-glucagon (Cat # 15954-1-AP,Proteintech Group, Inc)) overnight at 4 °C and incubated with appropriate fluorescent secondary antibodies (DyLight 488 Anti-Rabbit IgG and DyLight 594 Anti-Mouse, Vector Laboratories) for detection. Cell nuclei were stained with DAPI.
In the sections stained for insulin and glucagon, the borders of each pancreatic islet were identified morphologically and marked using the pen tool in ImageJ. The area of each islet was calculated with ImageJ software using the set scale function corresponding to the 20x scale bar. Insulin-positive nuclei were counted based on fluorescent signal. All islets with an area >566 μmˆ2 (corresponding to a diameter of >27 μm), approximately corresponding to islets containing a minimum of eight nuclei, were regarded for analysis.