Immunofluorescence Analysis of HESC-Derived Cells and Mouse Pancreas

HESC-derived cells and frozen sections of mouse pancreas were prepared and stained for immunofluorescence (IF) analysis using previously described protocols (26 (link),28 (link)). Primary and secondary antibodies are listed in Supplementary Table 1. Apoptotic cells were labeled using a TUNEL Assay Apoptosis Detection Kit (catalog no. 30064; Biotium). Entire sections of mouse pancreas or day 27 HESC clusters were imaged using a Zeiss LSM 710 confocal scope. HESC day 12 pancreatic progenitors were imaged using an Olympus IX53 fluorescent microscope. Fluorescently labeled cells and pancreas area were manually quantified using ImageJ software. Cells were quantified on at least three sections of mouse pancreas spaced 200 μm apart and on four sections of different day 27 HESC–derived clusters. Pancreatic progenitors were quantified on four random images in the well.