Immunofluorescence assay (IFA)

After dissection in 1xPBS, midguts, ovaries, and salivary glands at 7, 10, 14, and 21 dpi were placed on SuperFrost Plus microscope slides (Thermo Scientific, USA). Midguts, ovaries, and salivary glands were fixed in 4% paraformaldehyde for 15 min, rinsed with 1xPBS and then incubated for 15 min in 0.1% of Triton X-100. They were washed again with 1xPBS (3x5 min). The slides were drained and incubated for 60 min with mouse monoclonal [B1412huM] (Abcam, MA) as the primary antibody at the concentration of 1:1000 and then washed with 1xPBS (3x5 min). After washing, all slides were then incubated for 60 min with 1: 5000 Goat Anti-Mouse IgG H&L (Fluorescein isothiocyanate: FITC) (ab6785) (Abcam, MA) as the secondary antibody and then washed with 1xPBS (3x5 min). Finally, a drop of Prolong gold antifade (Invitrogen, USA) was applied on each slide, and a cover slide was placed on top. The negative mosquitoes run as a negative control. All slides were examined under a fluorescence microscope (Nikon, Japan).