Detection and visualization of VLP assembly and budding via electron microscopy. M, N, E, and S-APEX2 were coexpressed in HEK293 cells. Thirty hours posttransfection, cells were fixed and processed for imaging. TEM images of ultrathin sections of resin-embedded cells subjected to APEX2-DAB assay exhibit intense staining of the perinuclear endomembrane system (A, inset). This region exhibited localized stained clusters of vacuolar structures enmeshed with tubular compartments (A, indicated with white rings). Some of these stained vesicular-tubular structures are filled with stained VLPs (A, within red and orange boxes). At higher magnification, they appear as swollen tubular structures resembling ERGIC compartments (B and E, white arrowheads) filled with stained spherical VLPs. The spherical structures within these compartments (C and D, red arrowheads) resemble VLPs in size range and morphology and carry the APEX2 specific stain.