Goat anti mouse igg conjugated with fitc

The immunofluorescence assay was performed as described previously^{59 (link)}. Briefly, 1 × 10⁴ ZIKV-infected Vero cells were added to a multi-well glass slides in a MOI 0.1 at rt for 1 h. Cells were then fixed with acetone 80% solution (v/v) and incubated at − 20 °C for 30 min. After each step, wells were washed 3× with PBS 1X. Following incubation for 30 min with primary antibody (mouse immune sera, 1:500), goat anti-mouse IgG conjugated with FITC (1:750; Sigma) was added for 30 min. Immunofluorescence assay was performed using fluorescence microscopy (Olympus BX21) and the images were captured by CellSens software.

For immunofluorescence, ovaries were obtained from adult female ticks at 10 days after engorgement as described above, fixed with 4% paraformaldehyde in 0.2 M sodium cacodylate buffer, and dehydrated in a graded series of ethanol and embedded in paraffin. Sections (4 μm) were prepared and mounted on glass slides. Paraffin was removed from the sections with xylene and the sections were hydrated by successive 5-min washes with a graded series of 100, 80, 75, and 65% ethanol. The slides were treated with EDTA antigen repair buffer for 30 min at 37°C, washed with PBST and incubated with 2% bovine serum albumin (BSA; Sigma-Aldrich) in PBST for 30 min at room temperature. The slides were then incubated for 14 h at 4°C with primary antibodies against vitellin diluted 1:5–1:200 in 2% BSA mixture and developed for 30 min with goat-anti-mouse IgG conjugated with FITC (Sigma-Aldrich) (diluted 1:500 in 2% BSA mixture) after 3 washes in PBST buffer. The slides were incubated for 20 min with DAPI (1:200, 2% BSA dilution) after 3 washes in PBST buffer and mounted in Fluoromount Aqueous Mounting Medium (Sigma-Aldrich). Samples were observed and photographed with 10X objective, 1.0X zoom and the excitation of 488 nm (FITC) and 405 nm (DAPI) under a Zeiss inverted fluorescence microscope (Zeiss, Germany).

The protocol was the same as that of our previous report (19 (link)). Briefly, infected LMH cells on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.25% Triton X-100 for 5 min, washed with PBS, blocked with 2% BSA for 30 min, and incubated with mouse-anti-CAstV anti-serum (1:100) in PBS for 45 min at room temperature. The cells were washed in PBS, incubated with goat anti-mouse IgG conjugated with FITC (SIGMA, Shanghai, China) for 30 min at room temperature, and then stained with 10 μg/ml of Hoechst 33342 dye (SIGMA, Shanghai, China) at room temperature for an additional 10 min. The images were captured with a Leica SP2 confocal microscope.