Scanning Electron Microscopy of Cultured HCEnCs

HCEnCs were seeded on FNC-coated glass coverslips and maintained in M5-Endo for at least 48 hours. Following a four-day storage, coverslips were removed and fixed with neutrally buffered 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) at 4 °C for 4 hours. Alternatively, samples were placed at 37 °C for 48 hours recovery, and subsequently fixed. Samples were processed as previously described^{17 (link)}. Briefly, samples were post-fixed in 1% osmium tetroxide at room temperature for 1 hour. The samples were then dehydrated, critical point dried (BALTEC, Balzer, Liechtenstein) and mounted onto a metal stub using carbon adhesive tabs. Samples were sputter-coated for 160 seconds with a 22 nm layer of gold-phalladium alloy (BALTEC) and examined under a scanning electron microscope (Quanta 650FEG; FEI, Hillsboro, OR, USA).