HeLa cells were instantly frozen using a high-pressure freezing machine (High Pressure Freezer Leica EM ICE equipped with the Coral Life workflow; Leica Microsystems) and freeze-substituted into Agar 100 Epoxy resin (Agar Scientific). As fiducial markers, 15 nm of gold-conjugated Protein A (Cytodiagnostics, AC-15-05-10) was absorbed on both sides of 250 nm sections. Sections were post-stained with 2% UA and lead citrate. Dual axis tilt series were acquired with a Tecnai G2 20 transmission electron microscope operated at 200 kV and equipped with an Eagle 4k HS CCD camera (FEI) by using the Serial EM software. Images were recorded over a -55° to 55° tilt range on average with an angular increment 1°.
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Also, this feature is unstable and may not work properly, and checksums of downloaded files are not verified.
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