Immunohistochemistry of Insulin in Pancreas

For immunohistochemistry (IHC), sections of pancreas (4 µm) were stained with antibodies against insulin (Cell Signaling, Danvers, MA, USA) as described previously [5 (link)]. The slides were incubated in biotin-conjugated secondary antibody (IgG, 1:200 dilution) and avidin-biotin peroxidase (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h each. Negative controls were run in parallel without primary antibody in the incubation. The results of the immunohistochemical staining were captured by a microscope with an imaging capture system (Nikon NIS-Elements BR) and analyzed and calculated with ImageJ (Opensource Java Imaging Processing Program by NIH http://imagej.nih.gov/ij/).