Samples from SC-islets and human islets were chemically fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, supplemented with 2 mM calcium chloride at RT, for 2 h. After washing, the specimens were osmicated in the same buffer with 1% nonreduced osmium tetroxide on ice, for 1 h. Specimens were then washed and dehydrated in increasing concentration of ethanol and acetone, before gradual embedding into Epon. After polymerization over 18 h at 60 °C, a pyramid was trimmed on the location of the embedded cells. Ultrathin, 60-nm sections were cut using an ultramicrotome, picked on Pioloform-coated single-slot grids and poststained with uranyl acetate and lead citrate. Micrographs were acquired with a Hitachi HT7800 microscope operated at 100 kV using a Rio9 CMOS-camera. One or two SC-islets were chosen randomly for examination and 11–12 SC-islet beta cells were selected for imaging on the basis of characteristic features of beta-like granules. Cells for analysis from two differentiation experiments were selected based on the characteristics features of mature granules and images for α-cells and β-cells were collected with random systematic sampling: 3-5 images per cell, 4 cells per aggregate and 5 aggregates per sample. ER morphology was evaluated from the micrographs acquired at nominal magnification of 5,000X.

2022