Immunofluorescent Labeling of Pancreatic Sections

The conditions used for paraffin embedding pancreatic sections and immunofluorescence labeling were described previously (20 (link)). The following primary antibodies were used for immunostaining: guinea pig α-insulin (1:2,000; Linco Research), guinea pig α-glucagon (1:2,000; Linco Research), rabbit α-glucagon (1:2,000; Linco Research), rabbit α-MafB (1:10,000; Bethyl Laboratories), rabbit α-MafA (1:1,000, Bethyl Laboratories), rabbit α-Slc30a8 (1:1,000, Mellitech), and rabbit α-Glut2 (1:1,000; Chemicon). Species-matched secondary antibodies were used for immune detection (Cy5-conjugated donkey anti–guinea pig, Cy5-conjugated donkey anti-sheep, Cy2-conjugated donkey anti–guinea pig, Cy2-conjugated donkey anti-rabbit, Cy3-conjugated donkey anti-rabbit [all 1:500; Jackson ImmunoResearch Laboratories]). Cy3-conjugated tyramide signal amplification (1:400, PerkinElmer) was used for detecting MafB labeling with the biotin-conjugated donkey anti-rabbit secondary antibody (1:500; Jackson ImmunoResearch Laboratories). Nuclear counterstaining was performed using YoPro1 or DAPI (Invitrogen). Immunofluorescence images were acquired with a Carl Zeiss LSM510 confocal microscope.