Rabbit α mafa

Pancreata from 2- to 3-month-old wild-type and mutant mice were processed for paraffin and frozen sections. Immunohistochemical analysis was performed as described previously (Matsuoka et al., 2003 (link)). The primary antibodies used were guinea pig α-insulin (1:2,000; Dako), mouse α-glucagon (1:2,000; Sigma), rabbit α-MAFA (1:5,000; Bethyl Laboratories), rabbit α-MAFB (1:1,000; Bethyl Laboratories), rabbit α-CHRN-A4, A5, B2, and B4 (1:100; Alomone Labs), goat α-ADRA2A (1:100; Sigma), rabbit α-ADRA2A, rabbit α-TH (1:1,000; Sigma), goat α-VAChT (1:1,000; Millipore), and rabbit α-β-tubulin (1:5,000; Covance). The specificity of primary nicotinic receptor antibodies was assessed using absorption assays (Figure S2). Secondary antibodies used were Cy2-, Cy3-, and Cy5- conjugated α-guinea pig, α-mouse, α-goat, and α-rabbit (1:500; Jackson ImmunoResearch). Nuclear counter-staining was performed using DAPI (1:6,000; Invitrogen).

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.