10 nm gold particles

Small blocks of tissue (0.5×0.5×1 mm) containing secretory cavities at different developmental stages were fixed in a PBS solution (pH 7.2) containing 4% paraformaldehyde and 0.5% glutaraldehyde, and embedded in LR White resin (Sigma). A Leica EM UC7 microtome was used for cutting the sections to a thickness of 70–80 nm.
A nickel grid containing the sections was washed three times with a droplet of PBS-Tween buffer solution for 5 min, and then blocked with 1% BSA for 20 min. The grid was washed three times with PBST for 5 min each and was then floated on PBST containing the anti-CgCaN-specific polyclonal antibodies (primary antibodies, 1:10 v/v) and left to incubate for 3 h at 37 °C. The grid was again washed three times with PBST for 5 min each, then floated on PBST containing colloidal gold antibodies (secondary antibodies, 1:50, v/v; 10-nm gold particles; Sigma-Aldrich) and incubated for 1 h at 37 °C. It was then washed three times with PBST for 5 min each, and twice with d₂H₂O for 5 min each. The grid was then stained with uranyl acetate and lead citrate. Control samples were prepared in a similar manner. In control A, pre-immunization serum was used to replace the primary antibody, and in control B the primary antibody was replaced with PBS. The sections were examined and photographed using a Philips Fei-Tecnai 12 TEM.

Double-immunogold localization studies of PVY-NIb and HSC70 were performed based on the PCC estimation results. Two sets of antibodies were used for the analysis. The first set consisted of primary and secondary antibodies that were used for the detection of NIb (see Section 2.4) at 1:100 dilution. The second set included a primary mouse monoclonal antibody for detecting HSC70 (Enzo Life Sciences, New York, NY, USA), while the secondary antimouse antibodies were coupled to 10-nm gold particles (Sigma Aldrich, Warsaw, Poland) and used at 1:100 dilution. The embedded plant material (see Section 2.5) was cut into 100-nm sections with an ultramicrotome, placed on nickel grids, and treated as described by Otulak and Garbaczewska and Kozieł et al. [30 (link),43 (link)]. Then the grids were treated and labeled using the double-immunogold technique, as previously presented by Kozieł et al. [44 (link)]. Double-immunogold colocalization of NIb and HSC70 was analyzed using a 2 × 2 contingency table, as described by Mayhew [45 (link)]. Assessments in the 2 × 2 contingency table were performed separately for selected cell structures including vacuoles, nuclei, and chloroplasts using Fisher’s exact test, and the results were computed using GraphPad Software [46 ]. For more detailed analysis, we also calculated the odds ratio, as described by Mayhew and Lucocq [47 (link)].

Healthy, infected, or agro-infiltrated leaves were ground in 25 mM sodium phosphate buffer pH 7.4 and clarified at 4000 g for 5 min at 4°C. The 300 mesh formvar carbon-coated nickel grids were coated with polyclonal anti-GFLV antibodies and covered with clarified plant extracts. Following a saturation step with BSA 2% w/v, normal goat serum 10% v/v, Triton X100 0.05%, and 22.5 mM sodium phosphate buffer pH 7.4, the grids were further incubated with a mix of three homemade monoclonal anti-GFLV antibodies [17 (link)]. Immunogold labelling was performed using anti-mouse antibodies conjugated with 10 nm gold particles (Sigma-Aldrich, Saint-Louis, MO, USA). Three washes with sodium phosphate buffer preceded each step of the decoration procedure. Samples were negatively stained with a 1% ammonium molybdate solution prepared in the grinding buffer. For further analysis, a fixation step was performed before saturation, using a solution of 1% paraformaldehyde diluted in the grinding buffer. Observations were done on a Philips EM208 transmission electron microscope. Film-based photographs were acquired onto Kodak Electron Image Films SO-163 and developed. Photographs were scanned to obtain digital images.