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Electron Microscopy: Double Immunogold Labeling. Ultrathin sections cut from samples included in LR White resin (LRW) (see Materials and Methods) were placed on nickel grids and etched in aqueous 0.56 M sodium metaperiodate for 10 min. After thorough washing with distilled water, the grids were incubated in 1% BSA in TBS with Tween (TBST) (0.05 M Tris· HCl, pH 7.5/0.5 M NaCl/0.3% Tween 20) for another 10 min. Sections were then incubated for 1 h with 1:500 rabbit anti-virion-associated protein (VAP) coiled-coil antibody (1) and for 1 h in 20% goat anti-rabbit immunoglobulins conjugated to 6-nm gold particles (GAR 6, Jackson ImmunoResearch), both diluted in TBST/1% BSA. Extensive washing in TBST separated the incubations with the two antibodies. After successive washing in TBST, TBS (TBST without Tween 20) and distilled water, grids were dried and covered with formvar membrane. Subsequently, the opposite side of the grid was treated as described above by using 1:400 anti-MP coiled-coil antibody (obtained from a rabbit immunized with MP coiled-coil peptide), followed by 20% goat anti-rabbit immunoglobulins conjugated to 18-nm gold particles (GAR 18, Jackson ImmunoResearch).

To validate the double immunogold labeling results, we performed two tests to confirm the absence of any reaction between the GAR 18-conjugated anti-rabbit antibody with the rabbit anti-VAP antibody applied to the opposite side of the grid. After treatment with anti-VAP and GAR 6 (as described above), sections on the opposite side of the grid were either treated with polyclonal rabbit antibody anti-olive latent virus 1 movement protein (MP) or not treated at all with a primary antibody and then incubated with 20% GAR 18. In both cases, the lack of GAR 18 on grids confirmed the absence of unspecific cross-decoration (Fig. 8).

1. Jacquot, E., Geldreich, A., Keller, M. & Yot, P. (1998) Virology 242, 395-402.

Fig. 8. Validation of immunogold labeling assay on cauliflower mosaic virus (CaMV)-infected Brassica rapa leaf tissue. Grids were treated with rabbit anti-virion-associated protein (VAP) coiled-coil antibody and goat anti-rabbit immunoglobulins conjugated to 6-nm gold particles (GAR6) on one side and 18-nm gold particles (GAR18) on the opposite side. (A) Electron micrographs of ultrathin sections of infected tissue showing two neighboring cells traversed by a modified plasmodesma containing virus particles and part of an inclusion body (bottom left corner). (Scale bar: 200 nm.) Magnified images of the modified plasmodesma (B) and a section of the CaMV inclusion body (C) both contain virus particles. Six-nanometer gold particles label VAP.