Lowicryl resin

To analyze the granular ultrastructure, the cells were fixed with 1% glutaraldehyde and 1% PFA in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C. Specimens were then fixed in 2% osmium tetroxide for 60 min at 4 °C. Dehydration of the fixed samples was performed, and the samples were transferred to Lowicryl resin (Polyscience, Niles, IL, USA). Samples were then sectioned (60 nm) with an ultramicrotome (UltracutUCT, Leica, Wetzlar, Germany) and collected on nickel grids. Post-embedding immunogold labeling was performed for insulin and glucagon labeling using the rabbit anti-insulin (Abcam), mouse anti-glucagon (Abcam), 5-nm colloidal gold conjugated to goat anti-rabbit IgG (Sigma-Aldrich), and 9–11-nm colloidal gold conjugated to goat anti-mouse IgG (Sigma-Aldrich). Following immunogold labeling, the sections were double-stained with 2% uranyl acetate for 20 min and lead citrate for 10 min. The sections were then viewed using TEM.

To analyze the granular ultrastructure, the cells were fixed with 1% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C. Specimens were then fixed in 2% osmium tetraoxide for 60 min at 4 °C. Dehydration of the fixed samples was performed and the samples were transferred to Lowicryl resin (Polyscience, Niles, IL, USA). Samples were sectioned (60 nm) with an ultramicrotome (UltracutUCT, Leica, Wetzlar, Germany) and collected on nickel grids. Post-embedding immunogold labeling was performed for insulin and glucagon labeling using the rabbit monoclonal anti-insulin antibody (Abcam; ab181547), mouse monoclonal anti-glucagon antibody (Abcam; ab10988), 5-nm colloidal gold conjugated to goat anti-rabbit IgG secondary antibodies (Sigma), and 9–11-nm colloidal gold conjugated to goat anti-mouse IgG secondary antibodies (Sigma). Following immunogold labeling, the sections were double stained with 2% uranyl acetate for 20 min and lead citrate for 10 min. The sections were then viewed under a transmission electron microscope.