Immunoelectron Microscopy of Viral Antigens

Figures

• 

  Figure 1. Example of humidified chamber setup. (A) Schematic of humidified chamber used for immunogold staining protocol. Chamber should have a lid (1) to prevent evaporation. There should be a raised platform (2), on which a piece of Parafilm can be placed (3). Incubation of grids with droplets of antibodies and/or buffers should be performed atop the Parafilm. Next to the platform should be a reservoir of water (4) that provides humidity to the chamber. Be sure to keep the grids on the Parafilm away from the water reservoir. (B) Humidified chamber used for the experiments described in this article. Chamber is made of an empty pipet tip box, 1.5‐ml microcentrifuge tube storage box, Parafilm, and water.

• 

  Figure 2. Immunoelectron microscopy of Flublok (Sanofi Pasteur), a commercially available influenza vaccine from 2017 to 2018 containing hemagglutinin from H1N1, H3N2, and influenza B viruses. (A) Negative stain of vaccine stained with 3% PTA. Scale bar, 100 nm. (B) Vaccine labeled for H1N1 hemagglutinin using polyclonal rabbit antibody and 10‐nm gold‐conjugated goat anti‐rabbit antibody (Aurion). Note that the complexes are small enough that antibody binding appears to alter the size of the complex (red arrows), as the antibodies can also be visualized. Scale bar, 100 nm. (C) Enlarged region from panel B showing complex containing H1N1 hemagglutinin antigen with immunogold labels (red arrow) compared with an unlabeled complex lacking H1N1 hemagglutinin antigens (white arrow). Scale bar, 50 nm.

• 

  Figure 3. Immunoelectron microscopy of pandemic H1N1 influenza virus (A/California/04/09) produced in MDCK cells. (A) Negative stain of influenza virus stained with 3% PTA. (B) Virus labeled for hemagglutinin with C179 HA‐stem binding antibody and 10‐nm colloidal gold–conjugated goat anti‐mouse antibody. (C) Virus labeled for neuraminidase with polyclonal rabbit antibody and 25‐nm colloidal gold–conjugated goat anti‐rabbit secondary antibody. (D) Virus double labeled for hemagglutinin (10‐nm gold labels) and neuraminidase (25‐nm gold labels). Scale bar, 100 nm.

• 

  Figure 4. Troubleshooting errors when preparing immunogold‐labeled or negative‐stained TEM grids. (A) 25‐nm gold labels cannot fully label Flublok influenza vaccine antigens or fully surround the complexes compared with 10‐nm gold‐labels shown in Figure 1B, possibly due to steric hindrance from neighboring gold‐labeled antibodies. Nevertheless, the labeled complexes appear larger because primary antibodies were successfully able to bind (red arrows). (B) Nonspecific background labeling of pandemic H1N1 influenza A virus prevents successful interpretation of antigen presence. Red arrows indicate antibody aggregates, which may also limit data interpretation. (C) Even in conditions of poor‐contrast negative staining, the presence of Flublok antigens is evidenced by 10‐nm gold‐labeled H1N1 hemagglutinin. (D) Dense negative staining of pandemic H1N1 influenza A virus makes samples appear opaque; thus, interpretation of morphology is challenging. (E) Heavy staining can lead to broken carbon on the grid of pandemic influenza A virus. (F) Fractal‐like artifacts due to interactions between phosphate‐containing buffer and PTA negative staining solution make analysis of the Flublok sample…