Pancreatic Tissue Immunohistochemistry

Pancreata were fixed in 10% zinc formalin overnight and then transferred to 70% ethanol before being paraffin embedded and cut into 5-µm-thick sections. Tissue (one slide per animal) was incubated overnight with guinea pig anti-insulin (1:50, ab7842; Abcam) and rabbit anti-glucagon (1:1,000, 20076; Immunostar) antibodies. Immunoreactivity was detected by incubation for 1 h with anti–guinea pig-Alexa Fluor 568 and anti–rabbit-Alexa Fluor 488, and tissue was counterstained with DAPI to label nuclei. Images of each slide were acquired at 20× with an Olympus VS1000 fluorescence slide scanner and analyzed using Visiopharm software. Regions of interest were drawn around the entire pancreas section (total area) as well as each individual islet (islet area), as defined by insulin and/or glucagon immunoreactivity. The α- and β-cells were counted by hand from 10 average-sized islets per pancreas using ImageJ.