Immunohistochemistry of Pancreatic Tissues

We performed immunohistochemistry as described^{6 (link)}. We applied heart-perfused fixation with 4% paraformaldehyde (PFA). Pancreata were incubated in 4% paraformaldehyde (PFA) at 4 °C overnight, washed with ice-cold PBS three times, and placed in 30% sucrose overnight^{16 (link)}. Tissue was embedded in Tissue-Tek optimal cutting compound (Sakura Finetek), frozen on dry ice, and cut into frozen 5-μm sections. We applied antigen retrieval to detect transcription factors (Nacalai USA Inc.). We used primary antibodies to INSULIN (A056401-2; Dako; 1:1000), GLUCAGON (G2654; Sigma-Aldrich; 1:1000), PDX1 (ab47308; Abcam; 1:100), E-cadherin (61018; BD Biosciences; 1:100), REG1 (AF1657; R&D systems; 1:100), REG2 (AF2035; R&D systems; 1:100), REG3d (MAB5678; R&D systems; 1:100), NR5a2 (PPH2325; R&D systems; 1:100), KI67 (GTX16667; Genetex; 1:100), MAFA (IHC-00352; Bethyl Laboratories; 1:100), Cleaved Caspase-3 (9661; Cell Signaling; 1:100), and ALDH1A3 (NBP2-15339; Novus Biologicals; 1:100) and Alexa Fluor–conjugated goat serum as a secondary antibody (Jackson ImmunoResearch Laboratories and Molecular Probes). The images were captured using a Zeiss LSM 710 confocal microscope using a 20× objective and analyzed using ZEN.