Rhodamine avidin d

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)}