Formvar coated copper grids

Samples were processed for TEM imaging as described previously [40 (link)]. After fixation, the fixative was aspirated with a glass pipette, and samples were postfixed in 2% osmium tetroxide for one hour. Subsequently, samples were placed through a dehydrating series of graded concentrations of acetone. Dehydrated samples were impregnated overnight in a 1:1 mixture of acetone and araldite epoxy resin at RT. After impregnation, samples were embedded in araldite epoxy resin at 60 °C and monolayer samples were embedded in araldite according to the popoff method [41 (link)]. Ultrathin sections (0.06 μm) were mounted on 0.7% formvar-coated copper grids (Aurion, Wageningen, the Netherlands), contrasted with 0.5% uranyl acetate and a stabilised solution of lead citrate using a Leica EM AC20 (Leica). Samples were observed using a Philips EM 208 transmission electron microscope (Philips, Eindhoven, The Netherlands) equipped with a Morada Soft Imaging System camera with corresponding iTEM-FEI software (Olympus SIS, Münster, Germany).

Cells cultured on plastic Thermanox® coverslips were prepared for ultrastructural analysis as previously described [4 (link)]. Briefly, postfixation of cells fixed with 2% glutaraldehyde was performed with 2% osmium tetroxide in 0.05 M sodium cacodylate buffer (pH = 7.3) at 4°C for 1 hour. Dehydration of the samples was performed by ascending concentrations of acetone. The dehydrated samples were impregnated overnight in a 1 : 1 mixture of acetone and araldite epoxy resin at room temperature. After impregnation, samples were embedded in araldite epoxy resin at 60°C using the pop-off method. Embedded samples were cut in slices of 40-60 nm with a Leica EM UC6 microtome (Leica, Wetzlar, Germany) and transferred to 0.7% formvar-coated copper grids (Aurion, Wageningen, the Netherlands). The samples were contrasted with 0.5% uranyl acetate and a stabilized solution of lead citrate using a Leica EM AC20 (Leica). TEM analysis was performed with a Philips EM208 S electron microscope (Philips, Eindhoven, the Netherlands) equipped with a Morada Soft Imaging System camera with corresponding iTEM-FEI software (Olympus SIS, Münster, Germany).

Following fixation with 2% glutaraldehyde (Laborimpex, Brussels, Belgium) in 0.05M cacodylate buffer (pH 7.3; Aurion, Wageningen, the Netherlands) at 4°C, the fixative was gently aspirated with a glass pipette, and the cells were postfixed in 2% osmium tetroxide (Aurion) for 1 h. Subsequently, the cell-seeded coverslips were put through a dehydrating series of graded concentrations of acetone and embedded in araldite according to the popoff method (32 (link)). Ultrathin sections (0.06 μm) were mounted on 0.7% formvar-coated copper grids (Aurion), contrasted with 0.5% uranyl acetate and a stabilized solution of lead citrate (both from Laurylab, Saint-Fons Cedex, France), and examined in a Philips EM 208 transmission electron microscope (Philips, Eindhoven, The Eindhoven) operated at 80 kV. The microscope was provided with a Morada Soft Imaging System (SIS; Olympus, Tokyo, Japan) camera to acquire high-resolution images of the evaluated samples. The images were processed digitally with iTEM-FEI software (Olympus SIS).