Glucagon

Each rat was sacrificed at 16 and 22 weeks, and the liver and pancreas were removed and fixed in 10% formalin solution. We created paraffin tissue blocks that were then sliced into 5µm sections. The paraffin was removed, and the tissue was stained with hematoxylin and eosin. Immunostaining was performed using citrate buffer (pH 6.0) that was boiled twice for 5 min each, and then the samples were incubated at room temperature in goat serum for 20 min. Tissue slices were incubated at 4℃ for 16 min with primary insulin (Invitrogen, Rockville, MD, USA) and glucagon (Dako, Glostrup, Denmark) antibodies and then washed three times with Tris-buffered saline, pH 7.4. Secondary antibodies were stained using a Vectastain ABC Kit (Vector Labs, Burlingame, CA, USA), and biotinylated guinea pig antibodies were incubated with insulin for 1 h. Then, insulin was incubated for 1 h at room temperature with streptavidin Alexa Fluor 546 (Invitrogen), and glucagon was incubated for 1 h at room temperature with rabbit Alexa Fluor 488 (Invitrogen). After the tissue was washed three times with Tris-buffered saline, nuclear staining was performed with 4',6-diamidino-2-phenylindole, and the tissue was mounted with conjugated mounting medium (Dako). Then the slides were photographed with a fluorescence microscope (Olympus, Tokyo, Japan).