Goat anti glucagon

For immunofluorescence microscopy, frozen sections (6–8 μm) of transplanted cells and pancreas were prepared. For triple staining of insulin, glucagon, and somatostatin, the primary antibodies were mouse anti-human insulin (1:100; BioGenex, Fremont, CA), goat anti-glucagon and goat anti-somatostatin (1:200; Santa Cruz, Dallas, TX). The secondary antibodies were anti-mouse IgG, and anti-goat IgG (1:200; Vector, Burlingame, CA). The fluoresceins were fluorescein avidin D, AMCA avidin D and rhodamine avidin D (1:200; Vector). The staining procedure followed the fluorescein M.O.M kit (FMK–220, Vector). Between application of the primary antibodies against insulin and glucagon and glucagon and somatostatin, the avidin/biotin blocking kit (sp-200; Vector) was used, following the manufacturer’s instructions. All cells in 10 random fields were scored. Data were expressed as the number of positive cells per mm² of tissue.
For electron microscopy, tissue was fixed and processed as previously described.^{9 (link)} For insulin immunoelectron microscopy, a postembedding immunogold procedure was used. Tissues and cells were embedded in LR White (ProSciTech, Thurigawa, Australia) and labeling procedures were performed as previously described.^{9 (link)}