Ultraview ers spinning disc

For immunofluorescence microscopy, cells grown on glass coverslips were washed twice in cold PBS before fixation in 100% ice-cold methanol for 10 min at −20°C. Methanol was removed and samples were air-dried at room temperature. Fixed cells were incubated for 1 hr in blocking buffer (5% BSA in PBS). Samples were incubated with primary antibodies diluted in blocking buffer for 2 hr. After washing thrice with PBS, samples were incubated for 1 hr with Alexa Fluor 488-, 568-, or 647-conjugated secondary antibodies (Molecular Probes, Life Technologies) in the dark and subsequently washed three times with PBS for 10 min each. Nuclear DNA was stained with 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Sigma-Aldrich) for 10 min, washed with PBS, and rinsed with ultrapure water. Coverslips were mounted on microscopy slides using Fluoromount G (SouthernBiotech). Fluorescence images were acquired with a Nikon Ti Eclipse microscope and analyzed with the NIS-Element AR software package. For 3D reconstruction, 130-nm optical sections were acquired with a Nikon TE2000-E confocal microscope equipped with an Ultraview ERS spinning disc (PerkinElmer). Images were deconvoluted with Autoquant X3 software (Media Cybernetics) and 3D reconstruction was performed with Imaris 8 (Bitplane).

Huh7 cells stably expressing YFP-Sec61β and mito-mTurquoise2 were grown in 6-cm diameter dishes (Mattek) containing gridded coverslips and infected with DVs at a MOI of 1. Three days post infection, cells were washed twice and fixed by 20 min incubation with PBS containing 4% paraformaldehyde at room temperature. After several times washing with PBS, cells were examined with an Ultraview ERS spinning disc (PerkinElmer Life Sciences) on a Nikon TE2000-E inverted confocal microscope. Cells of interest were selected, and 0.13-μm optical sections in the cyan and YFP channels were acquired. The corresponding coordinates were also recorded using transmitted light with a differential interference contrast configuration. Cells were prepared and embedded for TEM analysis as previously described, and blocks were trimmed around the cell of interest according to the recorded coordinates. Ultrathin sections were prepared and examined with an EM-10 transmission electron microscope (Zeiss) containing a built-in MegaView camera (Olympus). Using ultrastructural and fluorescent patterns of mitochondria morphology and distribution, EM images and corresponding confocal microscopy Z-stacks were superimposed with the Photoshop CS5.1 software package (Adobe) and used for correlation analysis.