Immunofluorescent Staining of Pancreatic Islets

Immunofluorescent staining was performed as previously published^{35 (link)} with modifications to account for age of the embryos analyzed. Briefly, embryos were blocked in 4% Bovine Serum Albumin (Gemini Bio Products) for 90 minutes at room temperature followed by incubation with primary antibodies for 48 hours at 4°C. The following primary antibodies were used: guinea pig anti-insulin (Agilent; 1:200), mouse anti-glucagon (Cell Signaling Technology; 1:100), and chicken anti-GFP (Aves Labs; 1:500). For visualization, embryos were incubated overnight at 4°C with secondary antibodies (Alexa-488, Cy3, or Alexa-647 (Jackson Immunoresearch)); DAPI (Sigma; 1:1000) was used to visualize nuclei. Images were acquired with a Zeiss 710 confocal microscope. For each embryo, images were captured every 1 µm across the principal islet. Individual endocrine cell types were quantified using ImageJ. Statistical significance was determined by one-way ANOVA with multiple comparisons using Tukey’s post hoc test (Prism7, GraphPad).