Em 10 microscope

For transmission electron microscopy, kidney cortical tissues were cut into small pieces (1 mm³), fixed with 2.5% glutaraldehyde, and embedded in Epon 812 (Polysciences Inc., Warrington, PA). Conventional electron micrographs were obtained using an EM-10 microscope (Zeiss) operated at 60 kV. Absolute counting of total measurable glycogen particles was performed in 10 random electron microscopy fields of glomerular podocytes per mouse in 6 mice per group.

Kidney cortex samples were fixed in 2.5% glutaraldehyde. Then ultrathin sections were cut and mounted on a copper grid. Images were photographed using an EM-10 microscope (Zeiss) operated at 80 kV. For counting the number of podocyte foot processes, 10 ± 5 random electron microscopic fields of glomeruli per animal group were examined. The morphologic features were assessed by a single observer in a blinded manner.

Immunosorbent electron microscopy was used to visualize complex formation in plant extracts. Pioloform-coated nickel grids (Plano, Wetzlar, Germany) were floated on 40 μl anti-TMV or anti-His antibodies (diluted 1:100 in PBS) for 20–30 min at room temperature. The grids were washed with PBST (PBS plus 0.5% (v/v) Tween-20), and blocked with 40 μl 0.5% (w/v) BSA (British BioCell, Cardiff, UK) for another 20–30 min and washed again. Then, 40 μl of leaf extracts were added and incubated for 30–60 min before washing with PBST. VNPs were detected with primary anti-His or anti-TMV antibodies diluted 1:1000 in PBS and incubation overnight. Secondary 15 nm gold-conjugated GAM or GAR antibodies (British BioCell, diluted 1:100 in PBS) were used to decorate the particles by incubation for 2–4 h. After extensively washing with PBST, PBS, and deionized water, particles were contrasted with 1% (w/v) uranyl acetate. The grids were air-dried and analyzed by transmission electron microscopy using a Zeiss EM10 microscope (Carl Zeiss, Jena, Germany). The thermostability of purified TMV-ST/Cel12A nanoparticles was determined by heating for another 1 h at 50°C before labeling with the immunogold antibody pairs described above.

Pioloform-coated nickel grids (Plano, Wetzlar, Germany) were used for all preparations. For immunogold decoration the grids were incubated on a drop of systemically infected plant leaf extract for 30 min at room temperature, washed once with 20 drops of PBS containing 0.1% Tween-20 (PBST), and blocked with 0.5% bovine serum albumin (Sigma, Taufkirchen, Germany) in PBS for 20 min. After blocking, adsorbed hybrid particles were incubated for at least 2 h with the iLOV-specific antiserum described above (diluted 1:100), washed again and the particles were detected with a goat-anti-rabbit antibody conjugated to 15-nm gold particles (British BioCell, Cardiff, United Kingdom) for at least 2 h. Before staining with five drops of 1% uranyl acetate, the grids were extensively washed with PBST and twice with water. Transmission electron microscopy was carried out using a Zeiss EM10 microscope (Carl Zeiss AG, Jena, Germany).
Hybrid particles were captured from systemically infected leaf extracts by an αiLOV antibody. Therefore, the grids were incubated for 30 min on the iLOV-specific antibody, before washing and blocking with 0.5% BSA as described above. After another washing step the antibody was used to capture the TMV-iLOV particles from leaf extracts. The grids were washed and stained as described above.