Pancreatic tissue sections (6 μm) were cut from frozen OCT-embedded control (n = 7) and NE (n = 6) fetuses and prepared for histological evaluation as described previously (Cole et al. 2009 (link); Leos et al. 2010 (link)). Endocrine cells were identified with guinea pig anti-porcine insulin (1:500; Dako, Carpinteria, CA), mouse anti-porcine glucagon (1:500; Sigma-Aldrich), rabbit anti-human somatostatin (1:500; Dako), and rabbit anti-human pancreatic polypeptide (1:500; Dako). Immunocomplexes were detected with affinity-purified secondary antiserum conjugated to Cy2 (rabbit), Texas Red (mouse), or 7-amino-4-methylcoumarin-3-acetic acid (AMCA, guinea pig; Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500 in PBS with 1% BSA. Fluorescent staining was visualized on a Leica DM5500 microscope system and digitally captured with a Spot Pursuit 4 Megapixel CCD camera (Diagnostic Instruments, Sterling Heights, MI). Morphometric analysis was performed with ImagePro 6.0 software (Media Cybernetics, Silver Spring, MD). Positive areas were determined from 25 fields of view (FOV = 0.39 mm2) on two pancreas sections per animal separated by ≥ 120μm intervals (total area = 19.5 mm2; coefficient of variation between sections = 10.7%).