Cell Fixation and Embedding for TEM

The cells were fixed in a mixture of 1.6% paraformaldehyde and 2.5% glutaraldehyde prepared with a phosphate buffer (0.1 M, pH 7.4) for 1.5 h at room temperature as described by Szokoli et al. (2016) (link). Alternatively, 2.5% glutaraldehyde diluted with 0.1M cacodylate buffer (pH 7.4) was used as a fixative. The cells were washed in the same buffer containing 12.5% sucrose and postfixed in 1.6% OsO4 (1 h at 4°C). Then the cells were dehydrated in ethanol gradient followed by ethanol/acetone mixture (1:1), 100% acetone and embedded in Epoxy embedding medium (FlukaChemie AG, St. Gallen, Switzerland) according to the manufacturer’s protocol. The blocks were sectioned with a Leica EM UC6 Ultracut and ultrathin sections were stained with aqueous 1% uranyl acetate followed by 1% lead citrate. All samples were examined with a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan) or JEM-2000 (JEOL Ltd., Tokyo, Japan) at 90 kV.