Glucagon

Pancreatic tissue samples were fixed with 4% paraformaldehyde for 24 hours and embedded within paraffin. Paraffin sections of 7 µm were dewaxed in xylene, and incubated with 0.5% hydrogen peroxide in methanol for 0.5 hours to block endogenous peroxidase activity. After washing with phosphate-buffered saline, tissue samples were blocked using normal goat serum for 0.5 hours, and then incubated with primary antibodies for insulin (Millipore, Billerica, MA, USA), glucagon, Glut2, monocyte chemoattractant protein-1 (MCP1), ATF6α, and GRP78 (Santa Cruz Biotechnology) for 24 hours. The tissue samples were then incubated with secondary antibodies conjugated with fluorescent dye (Thermo Fisher Scientific) for 1 hour. DAPI (4′,6-diamidino-2-phenylindole) solution (Invitrogen, Carlsbad, CA, USA) was used to visualize the nucleus, and fluorescent images were obtained using an Olympus BX52 microscope (Olympus Optical Co., Tokyo, Japan).

Pancreatic tissues were fixed in 4% paraformaldehyde and embedded in paraffin. For IHC, 5-μm sections were stained with antibodies or control IgG using the EnVision+System-HRP (DAB) kit (Dako North America Inc, USA) according to the manufacturer’s instructions. For IF, tissues were incubated with antibodies against insulin (Abcam; Santa Cruz Biotechnology), glucagon (Santa Cruz Biotechnology), somatostatin (Santa Cruz Biotechnology), Zhx2 (Proteintech), PAX6 (Santa Cruz Biotechnology), and Ki67 (Abcam) overnight at 4°C and incubated with antibodies against Alexa Fluor 594 goat anti-guinea pig IgG (Abcam), Alexa Fluor 594 goat anti-mouse IgG (Proteintech), and Alexa Fluor 488 goat anti-rabbit IgG (Proteintech) for 1 h at 37°C. Microscopy analysis was performed with an Olympus IX51 microscope and ImageJ software.