Quetol spurr resin

Collected cell pellets were fixed by 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 hours at room temperature. After washing three times with the same buffer, the cells were serially dehydrated by 70% ethanol, 90% ethanol, and 100% ethanol solution at 10-min intervals. The dehydrated cells were embedded with Quetol-Spurr resin (EM0300, Sigma-Aldrich) overnight, sliced to 70- to 80-nm sections with an ultracut microtome (Ultracut RMC MT6000, Leica Microsystems), and loaded onto carbon-coated copper grids (Ted Pella). Cells were examined with a transmission electron microscope (FEI Tecnai 20, Thermo Fisher Scientific) using a 100-kV accelerating voltage.

Diluted samples were fixed by 4% PFA at RT for 10 min and labeled by anti–TRA-1-60 (dilution: 1:50; Miltenyi Biotec) in 1 ml of 1% BSA for 30 min at RT. Labeled samples were washed with 1% BSA in PBS twice and centrifuged at 2000 rpm for 4 min to form pellets. Pellets were then dehydrated with increasing concentrations of ethanol at 10-min intervals and embedded with Quetol-Spurr resin (Sigma-Aldrich) overnight. Samples were sliced to 70- to 80-nm-thick layers by an ultramicrotome (Ultracut RMC MT6000, Leica Microsystems, Germany) and deposited on electron microscopy grids (Ted Pella Inc.). Samples were observed under a transmission electron microscope (FEI Tecnai 20, Thermo Fisher Scientific) using 100-kV accelerating voltage.