Low viscosity resin

Hepatopancreases were removed from L. quadripunctata by dissection and fixed and dehydrated as described for fecal pellets used for SEM (s.a.) before infiltration with low viscosity resin (Agar Scientific) and polymerization at 60 °C for 16 h. For TEM, gold sections (70 nm) were cut using a Leica Ultracut UCT and mounted on 200 mesh copper grids. Sections were post-stained with 2% aqueous uranyl acetate (10 min) and lead citrate (5 min in a carbon dioxide-depleted chamber). Sections were viewed with an FEI Technai G2 TEM operating at 120 kV.

For electron microscopy specimen preparation, 80%–90% confluent Vero E6 monolayers in 25 cm² flasks were inoculated with stock virus and monitored for CPE. At the first sign of CPE, culture supernatant was collected, cleared of cellular content by centrifugation (3000× g for 5 min), and subsequently fixed in an equal volume of 2.5% glutaraldehyde in 0.1 M Hepes buffer (pH 6.9) for visualization of virus particles by negative staining. A Beckman Airfuge^® (Beckman Coulter, Brea, CA, USA) was used to concentrate all samples (10 min at 207 kPa), after which droplets of sample were adsorbed to 0.25% formar-coated copper grids for a minimum of 10 min, rinsed twice in deionised, distilled water and stained briefly in 2% phosphotungstic acid (pH 6.9). For ultramicrotomy, the remaining infected monolayers were flooded with the same fixative overnight, then routinely processed (postfixation in 1% buffered osmium tetroxide, graded ethanol dehydration, infiltration with a low viscosity resin (Agar Scientific, Stansted, UK) and overnight polymerisation at 70 °C). Seventy nm sections were cut on a Leica EM-UC6, double stained with saturated uranyl acetate and lead citrate, and viewed at 80 kV on a BioTwin Spirit (FEI Company, Hillsboro, OR, USA). Imaging was done with an Olympus Quemesa CCD camera (Olympus, Tokyo, Japan).

Samples were fixed with 4% PFA in PBS and send in the fixative. After several washing steps with PBS samples were further fixed with 1% osmiumtetroxide in PBS for 1 h at room temperature (RT). After a washing step with PBS samples were dehydrated with 10%, 30%, and 50% acetone on ice before incubation in 70% acetone with 2% uranylacetate for overnight at 7 °C. Samples were further dehydrated with 90 and 100% acetone on ice, allowed to reach room temperature and further dehydrated with 100% acetone, then changed into 100% ethanol. Subsequently, samples were infiltrated with the epoxy resin Low Viscosity resin (Agar Scientific, Stansted, UK). After polymerization for 2 days at 75 °C ultrathin sections were cut with a diamond knife, collected onto butvar-coated 3000 mesh grids, and counterstained with 4% aqueous uranylacetate for 4 min. Samples were imaged in a Zeiss TEM 910 at an acceleration voltage of 80 kV and at calibrated magnifications. Images were recorded digitally at calibrated magnifications with a Slow-Scan CCD-Camera (ProScan, 1024 × 1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Münster, Germany). Contrast and brightness were adjusted with Adobe Photoshop CS5 and afterwards sharpened with Image J Fiji (NIH, USA).

Transmission electron microscopy of V. album leaf cells was carried out as described previously (Senkler et al., 2018 (link)). In brief: Freshly harvested V. album leaves were cut into 1 mm pieces. The pieces were fixed in 150 mM HEPES, pH 7.35, containing 1.5% [v/v] formaldehyde and 1.5% glutaraldehyde [v/v] and washed with water. Afterward, the samples were incubated for 2 h in 1% [w/v] OsO₄ solution containing 1.5% [w/v] hexacyanoferrat II, subsequently washed with water and stored in 1% [w/v] aqueous uranyl acetate solution overnight. On the next day, the samples were washed again with water, dehydrated in acetone, and finally embedded in Low Viscosity Resin (Agar Scientific, Essex, UK). Ultrathin sections (60 nm) were mounted on formvar-coated copper grids and poststained with uranyl acetate and lead citrate (Reynolds, 1963 (link)). Samples were examined using a Morgagni Transmission Electron Microscope (FEI).

Fluorescence in situ hybridization using a combination of two Cy3-labeled probes (Chls-0523, E25-454; Thermo Fisher Scientific, Waltham, MA, USA) was performed as described elsewhere (35 (link), 80 , 81 (link)), and cells were stained with DAPI for 5 min. Images were recorded with a charge-coupled-device (CCD) camera (AxioCam HRc; Carl Zeiss) connected to an epifluorescence microscope and were processed using the AxioVision 4.6.3 software package (Carl Zeiss).
For transmission electron microscopy, the culture medium was replaced with fixative solution (2.5% glutaraldehyde in 3 mM cacodylate containing 0.1 M sucrose; pH 6.5). Amoebae were fixed for 1 h at room temperature, then collected, washed three times (0.1 M cacodylate containing 0.1 M sucrose [pH 7.2]), and mixed with one drop of 1% Biozym plaque agarose (Biozym, Hessisch Oldendorf, Germany) in washing buffer equilibrated at 35°C. Secondary fixation was conducted in 1% buffered osmium tetroxide for 1 h on ice, followed by dehydration in ethanol and infiltration with low-viscosity resin (Agar Scientific, Essex, United Kingdom). Ultrathin sections (70 nm) were cut using a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany) and stained with 0.5% uranyl acetate and 3% lead citrate, and imaging was done with a Zeiss EM 902 transmission electron microscope (Carl Zeiss).