Insulin and Glucagon Immunofluorescence in Pancreatic Islets

The primary polyclonal rabbit anti-insulin antibody was from Abcam (Cambridge, United Kingdom), and the primary polyclonal rabbit anti-glucagon antibody was from Dako (Carpinteria, CA, USA). The secondary antibody Alexa Flour 488 goat anti-rabbit IgG was from Life Technologies (Rockville, MD, USA). Poly-l-lysine, diazoxide, glutamic acid, and HEPES were from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Life Technologies-Gibco (Grand Island, NY, USA). The insulin and SSTR-2 antagonists S961 and PRL2903 were kind gifts from Novo Nordisk, Bagsværd, Denmark, and Dr. D. H. Coy (Tulane University, New Orleans, LA, USA), respectively. Adenovirus encoding the fluorescent ATP biosensor construct Perceval (26 (link)) was used according to a published method (29 (link)). Superfusion and batch incubation of islets were made with experimental medium containing 138 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl₂, 1.3 or 2.6 (hormone release) mM CaCl₂, 3 mM glucose, 0.5 mg/ml BSA, and 25 mM HEPES with pH adjusted to 7.4 with NaOH.