The fixed tissues were dehydrated with a graded ethanol series, cleared with xylene, and embedded in paraffin. For general morphology, serial horizontal sections (5 μm in thickness) were cut, deparaffinized, and stained with hematoxylin and eosin. The dorsal pancreas was identified as the organ in the dorsal mesogastrium connecting the duodenum and spleen, located dorsal to the stomach. For immunohistochemistry, sections were blocked with 0.3% hydrogen peroxidase in methanol (20 min) to inhibit endogenous peroxidase activity, treated with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS, 30 min) to block nonspecific antibody binding, and processed as described previously (Hayashi et al.,2003). The following primary antibodies were diluted with PBS containing 1% BSA and applied to the sections: rabbit polyclonal anti-glucagon antibody (1:500; Zymed Labs, Co., San Francisco, CA), guinea pig polyclonal anti-insulin antibody (1:1,000; DAKO, Carpinteria, CA), rabbit polyclonal anti-cytokeratin wide spectrum screening antibody (1:100; DAKO), and goat polyclonal anti-secretogranin III antibody (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For enzymatic detection of the bound primary antibodies, alkaline phosphatase (ALP) -conjugated anti-rabbit IgG, horseradish peroxidase (HRP) -conjugated anti–guinea pig IgG (both from Chemicon, Temecula, CA), and biotinylated anti-rabbit IgG and biotinylated anti-goat IgG (both from Vector Laboratories, Burlingame, CA) were used at 1:200 dilution (PBS with 1% BSA). For biotinylated antibodies, the sections were further incubated with HRP-conjugated streptavidin (1:500, Vector Laboratories). Enzyme reaction was detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB) for HRP, and Vector blue ALP substrate (Vector Laboratories) for ALP. For immunofluorescence, Alexa Fluor 568–conjugated anti-rabbit IgG, and Alexa Fluor 488–conjugated anti-mouse IgG (both from Molecular Probes, Eugene, OR) were used at 1:200 dilution (PBS with 1% BSA). Sections were observed under a microscope (AX70, OLYMPUS, Tokyo, Japan). For double immunostaining for glucagon and insulin, both antibodies were simultaneously applied on sections. For negative control, sections were incubated with 1% BSA in PBS instead of the primary antibody and treated similarly. No significant reactivity other than nonspecific autofluorescence of erythrocytes was observed in all the negative controls.