1 k fastscan ccd camera

Cells were fixed in culture dishes with 2.5% glutaraldehyde in Seahorse XF DMEM medium (Agilent Technologies; pH 7.4) for 1 h at RT and washed once in Seahorse XF DMEM medium. Cells were harvested by scraping, pelleted for 5 min at 2000 rpm at RT and washed two times in cacodylate buffer (100 mM, pH 7.4). Cell pellets were post-fixed with 1% osmium tetroxide in cacodylate buffer for 2 h, dehydrated in an ascending ethanol series and stained with 2% uranyl acetate in 50% ethanol. The samples were embedded in Araldite resin (Plano) according to the manufacturer's instruction. Ultrathin sections of 70 nm thickness were cut using an ultramicrotome Ultracut E (Reichert-Jung) and mounted on Formvar carbon-coated 100 mesh grids (Quantifoil). The Ultrathin sections were stained with lead citrate for 10 min [19 (link)] and examined in a Zeiss CEM 902 A transmission electron microscope (Carl Zeiss). Images were acquired using a 1 k FastScan CCD camera (camera and acquisition software, TVIPS).

Livers were perfused, cut into pieces, fixed and washed as described for scanning EM (see above).
For the analyses of tight junction functionality, mouse livers were additionally perfused through the inferior vena cava with 3% (w/v) lanthanum nitrate in 4% (w/v) PFA for 10 min^{28 (link)} after short-term perfusion with 4% (w/v) PFA for 1 min.
The samples were prepared according to procedures described before^{70 (link)}. In brief, for contrasting, liver samples were incubated with 1% (w/v) OsO₄ for 2 h and washed three times with 0.1 M sodium cacodylate buffer. Samples were then dehydrated by rising ethanol concentrations and stained with 2% (w/v) uranylacetate in 50% (v/v) ethanol for 1 h before they were embedded in araldite resin at 60 °C for 48 h.
After ultrathin sectioning of the embedded samples using a LKB 8800A Ultratome III (LKB Produkter AB), the sections (60 nm) were placed on formvar-coated grids and were finally stained with 3% (w/v) lead citrate in ddH₂O (Electron Microscopy Sciences) for 2 min.
The sections were investigated in an EM902A transmission electron microscope (Carl Zeiss AG) operated at 80 kV and images were recorded with a 1 k FastScan CCD camera (TVIPS camera and software).

Selected bacterial strains were grown overnight in LB medium at 37 °C with shaking at 200 r.p.m. In the case of mitomycin-C-treated cultures, mitomycin C was added in late exponential phase to a final concentration of 0.5 μg ml⁻¹. Culture supernatants were collected, mixed at a 1:4 ratio with PEG-8000 solution (PEG-8000 20%, 2 M NaCl), incubated on ice for at least 90 min and finally centrifuged (20 min, 7,600 r.p.m.) to obtain precipitate. The pellet was resuspended in 10% of the original supernatant volume in TBS solution (50 mM Tris-HCl, 150 mM NaCl, pH 7), incubated on ice for 90 min and centrifuged (20 min, 7600, r.p.m.). Supernatant was carefully transferred to clean Eppendorf tubes. Purified samples (100 μl) were adsorbed onto duplicate 400 mesh carbon-coated Cu grids (Quantifoil, Großlöbichau, Germany) for 2 min. Before use, the carbon grids were hydrophilized by 30 s of electric glow discharging. The grids were washed twice in distilled water and stained for 30 s with 1% uranyl acetate. Virus morphologies were examined using a Zeiss CEM 902A transmission electron microscope (Carl Zeiss AG, Oberkochen, Germany). At least 20 images were taken per sample at different magnifications using a 1k FastScan CCD-Camera (camera and software from TVIPS, Munich, Germany).