Transfer of Anti-Rotavirus Antibodies during Pregnancy and in Milk Following Maternal Vaccination with a Herpes Simplex Virus Type-1 Amplicon Vector

Meier, Anita F.; Suter, Mark; Schraner, Elisabeth M.; Humbel, Bruno M.; Tobler, Kurt; Ackermann, Mathias; Laimbacher, Andrea S.

The grids are recovered from the fluorescent microscope settings and further processed for electron microscopy. The ultrathin sections were stained with uranyl acetate and lead citrate. Finally, the grids are loaded into the scanning transmission electron microscope (STEM) holder of the SEM (Helios NanoLab 650, FEI Company, Eindhoven, The Netherlands). The images taken with the fluorescence microscope were opened on the SEM computer using the Maps software (FEI Company, Eindhoven, The Netherlands), a specially designed software to correlate light and electron microscopy which can read any type of images obtained by any type of light microscope. An electron micrograph was recorded using the Everhart-Thorney secondary electron detector and the alignment between the light and the electron micrograph was done using the Maps software using two auspicious points. A grid of image tiles, tileset, was drawn over the area of interest, indicated by the fluorescence image. The tile size was determined by the imaging conditions and had an overlap of 10%. Imaging conditions were: 30 keV, 1.6 nA, 5 mm working distance, 6144 × 4096 pixels per frame, 5 µm horizontal field of view (about 8 Å pixel size), 1 µs dwell time using the STEM III detector (FEI Company) in the high-angle annular-dark field mode. We used the three-point focus regime: at three points close to the area of interest the section was focused at higher magnification, then brightness and contrast were adjusted at the magnification and imaging conditions used for recording the images. Finally, the tiles were stitched by the software and exported as tiff files. To circumvent any disturbing effects (e.g., bleaching), occurring in the overlapping zones after stitching, the whole area of interest was pre-irradiated with a high current (26 nA), large frame size (6144 × 4096 pixels) and short dwell time (50 ns) for about 30 min.