TEM analysis and IFA of fusion-GEM complexes
For transmission electron microscopy (TEM) observation, the fusion-GEM
complexes were centrifuged to obtain the precipitate. After fixation
with 2% glutaraldehyde (no suspension) overnight at 4°C, the
precipitate was sliced into ultrathin sections. Then, the samples were
stained with uranium acetate and lead citrate, and their features were
observed under a transmission electron microscope.
For the IFA, the precipitate from centrifuged fusion-GEM complexes was
suspended and blocked with 5% nonfat milk. Then, the cells were stained
with an anti-flavivirus E mAb (1:200) as the primary antibody and with
FITC anti-mouse IgG (1:200) as the secondary antibody. The stained
complexes were applied to slides and observed under a fluorescence
microscope (Olympus Corp, Tokyo, Japan).