Alexa fluor 488 green affinipure goat anti mouse igg

Midguts or salivary glands from viruliferous, non-viruliferous or dsRNA-treated planthoppers were dissected in 1× PBS buffer (pH 7.2) on a glass plate and fixed in 4% (w/v) paraformaldehyde for 2 h at room temperature. After being treated with osmotic buffer (0.01 mol/L phosphate-buffered saline containing 2% Triton X-100, pH 7.4) for 2 h, the tissues were incubated with the monoclonal anti-NP antibody and/or the polyclonal anti-importin α3 antibody overnight at 4 °C. After being washed with 0.01 mol/L phosphate-buffered saline containing 2% Tween-20 (pH 7.4), the tissues were blocked with 3% bovine serum albumin for 1 h. Then, the secondary antibody Alexa Fluor 488 (green) affinipure goat anti-mouse IgG or Alexa Fluor 594 (red) affinipure goat anti-rabbit IgG (YEASEN) was added. The nuclei were labeled with Hoechst (blue). The samples without the treatment of primary antibodies were used as negative controls. The images were viewed under a Leica TCS SP5 confocal microscope (Leica Microsystems, Solms, Germany).

The NP ORF was cloned into the plasmid pAc-5.1/V5-HisB (Invitrogen). The C2 fragment of NP (204 to 284 aa) and GFP were cloned into the plasmid pIEx-4 between the restriction sites KpnI and NotI to yield the plasmid pIEx-4-GFP-Gly-Ser (linker)-C2. The primers used for cloning are listed in Table S3, and the plasmids pAc-5.1/V5-HisB and pIEx-4-GFP were used as controls. For each plasmid, 20 ng was transformed into Drosophila S2 cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific). The cells were sampled at 45 h after transfection and fixed with 4% (w/v) paraformaldehyde for 30 min. For NP-expressing cells, the monoclonal anti-NP antibody and Alexa Fluor 488 (green) affinipure goat anti-mouse IgG (YEASEN, Shanghai, China) were sequentially added. For GFP- and GFP-C2-expressing cells, GFP signals were directly observed. Nuclei were labeled with Hoechst (blue), and fluorescence was examined under a Leica TCS SP5 confocal microscope (Leica Microsystems, Solms, Germany).