Orius sc200 ccd camera

A 2-mm region at the base of stems at growth 6.9 were chemically fixed, dehydrated, and embedded in LR white according to the method outlined in Wilson and Bacic (2012) (link). Thin sections (∼80 nm) were acquired for antibody labeling and post-stained (Wilson and Bacic 2012 (link)). For antibody labeling, samples were incubated with anti-6x-His tag monoclonal (Invitrogen, # MA1-21315) at 1:100 dilutions for 1 h at room temperature, and then overnight at 4 °C. Samples were then washed and incubated with goat anti-mouse 18 nm gold-conjugated secondary antibody (Jackson Immuno Research #115-215-166) at 1:20 dilutions for 1 h at room temperature. Detection of ultrastructure, HIS-YFP-FLA11, and HIS-YFP-FLA12 subcellular location was performed on 2 biological replicates using 2 technical replicates. Grids were imaged using a Jeol 2100 EM equipped with a Gatan Orius SC 200 CCD camera. Gold signals were quantified manually from TEM images and data were shown as mean ±Sd.

Analysis of samples containing outer membrane vesicles by dynamic light scattering (DLS) was performed using commercial service at SOLVE analytical laboratory (Lund, Sweden). OMV containing samples were analysed using Zetasizer Nano ZS instrument (Malvern, UK) and Zetasizer Software 8.01 (Malvern, UK). Transmission electron microscopy (TEM) was done at BioVis core facility at Uppsala University (Sweden). A 5 µl drop of the sample was placed on a Formvar- and carbon coated 200-mesh copper grid. The excess solution was immediately removed by blotting with filter paper. The sample was then washed on three consecutive drops of MQ water followed by two drops of 2% uranyl acetate. Excess of uranyl acetate was removed by blotting on filter paper. Dried grids were examined by Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher/FEI) at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

Samples were fixed in 2.5% glutaraldehyde (Ted Pella) + 1% paraformaldehyde (Merck) in PIPES pH 7.4 and stored at 4°C until further processed. Samples were rinsed with 0.1 M PBS for 10 min prior to 1 h incubation in 1% osmium tetroxide (TAAB; Berks, UK) in 0.1 M PBS. After rinsing in 0.1 M PBS, samples were dehydrated using increasing concentrations of ethanol (50%, 70%, 95% and 99.9%) for 10 min each step, followed by 5 min incubation in propylene oxide (TAAB). The samples were then placed in a mixture of Epon Resin (Ted Pella) and propylene oxide (1:1) for 1 h, followed by 100% resin and left overnight. Subsequently, samples were embedded in capsules in newly prepared Epon Resin and left for 1–2 h and then polymerised at 60°C for 48 h. Ultrathin sections (60–70 nm) were cut in an EM UC7 Ultramicrotome (Leica) and placed on a grid. The sections were subsequently contrasted with 5% uranyl acetate and Reynold's lead citrate and visualised with Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher/FEI) at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

The original sample, selected test plants (see “First Test on Sample HMV-SI/L”) and leaves from S. lycopersicum and N. benthamiana (Figure 1B) infected with the HMV-SI/L isolate were examined using TEM. The sample (20 μl) was applied to Formvar-coated, carbon-stabilized copper grids and negatively stained using a 1% aqueous solution of uranyl acetate (SPI Supplies), followed by visualization using Philips CM 100 transmission electron microscope (FEI, Eindhoven, Netherlands). Images were captured using an ORIUS SC 200 CCD camera (Gatan Inc., Pleasanton, United States).

EV samples were mixed with an equal volume of 4% paraformaldehyde, added onto a formvar and carbon-coated 200-mesh grid (Oxford 11 Instruments) and incubated for 20 min. After incubation, the grid was dried and washed first 3 times with PBS, followed by 8 washes in MilliQ water. The samples were stained in a drop of uranyl oxalate, pH = 7, for 5 min, after which they were stained with a drop uranyl acetate 4% pH = 4 with 2% methylcellulose on ice for 10 min. The dried grids were imaged in a Tecnai G2 transmission electron microscope (TEM, FEI company) with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

Characterization of Tau-F, Tau-O and BDTau-F was performed by transmission electron microscopy (TEM) using negative staining. Tau-F were diluted 1:10 in distilled H2O and placed on a formvar and carbon coated 200-mesh copper grid (Ted Pella). The sample was directly stained with 2% uranyl acetate. Dried grids were examined by TEM (FEI Tecnaii G2) operated at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).