Durcupan epoxy resin

Mice were perfused with 10ml of modified Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4) carefully. Liver were then dissected and fixed for at least 4 hours, postfixed in 1% osmium tetroxide in 0.15 M cacodylate buffer for 1 hour and stained en bloc in 2% uranyl acetate for 1 hour. Samples were dehydrated in ethanol, embedded in Durcupan epoxy resin (Sigma-Aldrich), sectioned at 50 to 60 nm on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 2% uranyl acetate for 5 minutes and Sato’s lead stain for 1 minute. Grids were viewed using a JEOL 1200EX II (JEOL, Peabody, MA) transmission electron microscope and photographed using a Gatan digital camera (Gatan, Pleasanton, CA), or viewed using a Tecnai G2 Spirit BioTWIN transmission electron microscope equipped with an Eagle 4k HS digital camera (FEI, Hilsboro, OR).

Early (day 5) and late stage (day 21) differentiating hESCs were generated as described above. Cell samples were prepared for imaging at the UCSD Cellular and Molecular Medicine Transmission Electron Microscopy Facility (http://emcore.ucsd.edu/TEM.html). Attached cell cultures (day 21) and EB suspension–cultured cells (day 5) were fixed using 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 min at room temperature before incubating on ice for 30 to 60 min. Cells were pelleted by gently scraping attached cells from petri dishes into 2% agarose (Sigma-Aldrich). EB suspension cultures were allowed to settle down using gravitational force before transferring them into 2% agarose. Pellets were fixed again using 2% glutaraldehyde [in 0.1 M sodium cacodylate buffer (pH 7.4)] for at least 4 hours, postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour, and stained en bloc in 2% uranyl acetate for 1 hour. Samples were dehydrated in ethanol, embedded in Durcupan epoxy resin (Sigma-Aldrich), sectioned at 55 nm on a Leica Ultracut UCT ultramicrotome, and picked up on copper 300-mesh grids. Sections were stained with 2% uranyl acetate for 5 min and with Sato’s lead stain for 1 min. Grids were viewed using a JEOL 1200EX II (JEOL) transmission electron microscope and photographed using a Gatan digital camera (Gatan).

EM was performed at the CMM Electron Microscopy Facility at University of California San Diego. Four-month organoids were immersed in modified Karnovsky’s fixative (2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.15 M sodium cacodylate buffer, pH 7.4) for at least 4 hours, post fixed in 1% osmium tetroxide in 0.15 M cacodylate buffer for 1 hour and stained in 2 % uranyl acetate for 1 hour. Samples were dehydrated in ethanol, embedded in Durcupan epoxy resin (Sigma-Aldrich), sectioned at 50 to 60 nm on a Leica Ultracut UCT (Leica, Bannockburn, IL), and transfer onto Formvar and carbon-coated copper grids. Sections were stained with 2 % uranyl acetate for 5 minutes and Sato's lead stain for 1 minute. Grids were analyzed using a JEOL 1200EX II (JEOL, Peabody, MA) transmission electron microscope equipped with a Gatan digital camera (Gatan, Pleasanton, CA).