Goat anti glucagon α cell

Paraffin-embedded pancreases were sectioned at 7-μm intervals and subjected to TUNEL staining using an in situ cell death detection kit (Roche Diagostics, Indianapolis, IN, USA). For islet cells, this reaction was blocked with a blocking buffer, and the slides were immunostained with rabbit anti-insulin (β cell) and goat anti-glucagon (α-cell; Cell Signaling Tech, Danvers, MA, USA) for 2 h and then with donkey-derived secondary antibodies conjugated to PE (Invitrogen, Carlsbad, CA, USA). All slides were mounted with 4′,6-diamidino-2-phenylindole-containing medium. Fluorescent slides were viewed using a Leica DM6000 microscope, and images were acquired using software. Apoptosis was analysed by counting the TUNEL-positive β cells in each islet. Alternatively, islets were isolated from pancreases, β cells in islets were gated as insulin positive cells with a flow cytometer and apoptotic cells were identified as TUNEL-positive cells.