Visualization of a luteovirus in the vector aphid's body by two gold immunolabelling techniques: a comparative study.
Two gold immunolabelling techniques using electron microscopy were compared to examine the in situ localization of a luteovirus, potato leafroll virus (PLRV), inside its main aphid vector, Myzus persicae SULZ. With Gildow's technique, virus particles were labelled prior to fixation, embedding by injecting PLRV-specific IgGs into the living aphids. This facilitated the detection of extracellular particles located between the basal lamina and plasmalemma by trapping them in aggregates. The heavy coating of particles by antibodies and gold indicated good labelling sensitivity. Isometric virus-like particles were also observed inside the cytoplasm, but they were non decorated because the cell membrane prevented labelling reagents from entering the cell. With the second technique, ultrathin sections were immunolabelled after fixation-embedding. Since PLRV lost its antigenicity when aphid tissues were normally treated for electron microscopy, the successful application of this technique required fixation in 4% formaldehyde before embedding in Lowicryl at low temperature; it was also necessary to use PLRV-specific monoclonal antibodies to eliminate non-specific reactions. In these conditions, all intra- and extra-cytoplasmic virions present on the surface of sections were surrounded by gold particles, but the antibody coating was not discernible, and, because the resin limited the access of markers to antigens, the inner virus particles were not labelled. In conclusion, both techniques must be applied on the same material to give complementary information.
Garret
A
A
Station de Pathologie Végétale, INRA, Centre de Rennes, France.
Kerlan
C
C
Thomas
D
D
D000180:Acrylic Resins; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D001042:Aphids / Q000033:anatomy & histology / Q000382:microbiology*; D004853:Epoxy Resins; D007150:Immunohistochemistry / Q000379:methods*; D016253:Microscopy, Immunoelectron; D010942:Plant Viruses / Q000276:immunology / Q000302:isolation & purification / Q000648:ultrastructure*; D011198:Solanum tuberosum / Q000382:microbiology*; D016610:Tissue Embedding; D014021:Tissue Preservation
D000180:Acrylic Resins; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004853:Epoxy Resins; C004875:EPON; C037883:Lowicryl 4KM resin