Second-instar N. lugens nymphs were inoculated on RRSV-infected rice
seedlings for 48 h and then transferred to healthy rice seedlings.
The salivary glands were dissected from the N. lugens nymphs at 48-h
intervals, and uninfected nymphs were used as controls. The salivary gland
samples were fixed using 4% paraformaldehyde in PBS for 6 h at
4 °C and then blocked with 10% fetal bovine serum
(Gibco) at room temperature for 2 h. A monoclonal antibody against a
major RRSV capsid P8 protein was diluted 1:200, followed by overnight incubation
at 4 °C and visualization using a FITC-labeled secondary
goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), diluted
1:100. After the subsequent washes, the tissues were stained using
100 nM DAPI (Sigma-Aldrich) for 2 min at room
temperature. Following three washes with PBS, each for 10 min,
fluorescence images were observed using a Zeiss LSM 780 confocal microscope
(Zeiss).
seedlings for 48 h and then transferred to healthy rice seedlings.
The salivary glands were dissected from the N. lugens nymphs at 48-h
intervals, and uninfected nymphs were used as controls. The salivary gland
samples were fixed using 4% paraformaldehyde in PBS for 6 h at
4 °C and then blocked with 10% fetal bovine serum
(Gibco) at room temperature for 2 h. A monoclonal antibody against a
major RRSV capsid P8 protein was diluted 1:200, followed by overnight incubation
at 4 °C and visualization using a FITC-labeled secondary
goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), diluted
1:100. After the subsequent washes, the tissues were stained using
100 nM DAPI (Sigma-Aldrich) for 2 min at room
temperature. Following three washes with PBS, each for 10 min,
fluorescence images were observed using a Zeiss LSM 780 confocal microscope
(Zeiss).