Tecnai g2 20 transmission electron microscope

Transmission electron microscopy of macrophages was performed as recently described [22 (link)]. Briefly, macrophages were cultured on an Aclar film, fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% formaldehyde (2 h), post-fixed in 2% OsO₄ (2 h), dehydrated in graded series of ethanol, and embedded in a TAAB epoxy resin.
For high-pressure freezing (HPF), macrophages were grown on carbon-coated sapphire discs and frozen under liquid nitrogen conditions using 2000 bar within ms. Freezing was followed by freeze substitution in acetone by adding 2% OsO₄ and 0.2% uranyl acetate at temperatures below −70 °C. After water in form of ice within the cells was replaced by substitution media, samples were embedded in epoxy resin.
Images of 75 nm sections (stained with lead citrate and uranyl acetate) were taken on a FEI Tecnai G2 20 transmission electron microscope (FEI, Eindhoven, Netherlands) with a Gatan ultrascan 1000 CCD camera (acceleration voltage 120 kV).

Cell cultures were fixed for 24 h at 4 °C by immersion in a fixative solution containing 3% glutaraldehyde in PBS buffer (0.1 M phosphate, pH 7.5) and then stored in PBS buffer at 4 °C until further processing. Samples were subsequently post-fixed for 1 h in a solution containing 1% osmium tetroxide in PBS (1×, final pH 7.5), washed with MilliQ water and dehydrated in an increasing gradient of ethanol before infiltration and embedding in Spurr resin (ProsciTech). Resin blocks were then cut into 90 nm sections using an Ultracut UC6 microtome (Leica Microsystems). Selected sections containing cells and EFVs were stained on finder grids (Electron Microscopy Sciences) with uranyl acetate and lead citrate. Stained sections on finder grids were viewed at 200 kV accelerating voltage using a FEI Tecnai G2 20 transmission electron microscope at the Mark Wainwright Analytical Centre: Electron Microscope Unit (University of New South Wales).