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 above described ultrathin sections of transduced HepG2 cells embedded in LR White were incubated for 20 min with 50 mM Glycine before blocking for 30 min with blocking buffer (0.5% BSA/0.1% gelatin (Cold Water Fish Skin, EMS, Hatfield, PA, USA)) followed by 5 min incubation in 0.1% acetylated BSA (BSA-c) (Aurion, Wageningen, The Netherlands), pH 7.5 at room temperature (RT). Incubation was done overnight with the polyclonal goat anti-RV serum (obtained from Catherine Eichwald, University of Zurich, Zurich, Switzerland) diluted 1:10 in 0.1% BSA-c at 4 • C. After washing five times with BSA-c, the sections were incubated with the secondary anti-goat antibody conjugated with Alexa Fluor 488 (Molecular Probes), diluted 1:500 in 0.1% BSA-c for 1 h at RT. After washing 5 times with 0.1% BSA-c and once with H 2 O, the sections were stained with DAPI (0.1 mg/mL) for 15 min followed by 2 washes with PBS. After the staining, the sections were sandwiched between a microscope slide and a coverslip in PBS and sealed with nail polish. With a fluorescence microscope (Axio Observer inverted microscope), the whole grid was imaged in bright field and fluorescence and several images were taken with a 100× oil (NA = 1.25) objective. These images served as maps and were then used to find the same areas in the scanning electron microscope (SEM).