Immunohistochemical analysis of pancreatic insulin

Rat Pancreas was fixed in 10% buffered neutral formalin then washed, dehydrated in ascending grades of ethyl alcohol then cleared in xylene, embedded in paraffin, sectioned at 5 micron thickness, deparaffinized, and then treated with 3% H2O2 for 10 min to block the peroxidases activity. Subsequently, the tissue samples were heated at 121°C in 10 mM citrate buffer for 30 min, blocked for 20 min in 5% normal serum. The pancreatic tissue was incubated overnight with rabbit polyclonal anti-insulin primary antibody (1:100; sc-9168; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or with GLUT4 Antibody (1:100; sc-53566; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in phosphate-buffered saline (PBS) at 4°C. After three extensive washes with PBS, the sections were incubated with a goat anti-rabbit IgG biotin conjugated secondary antibody (1:2,000; sc-2040; Santa Cruz Biotechnology, Inc.) for 20 min at 32°C. After further incubation with horseradish peroxidase-labeled streptavidin, antibody binding was visualized using diaminobenzidine, and the sections were counterstained with hematoxylin.