Prolong gold antifade containing 4 6 diamidino 2 phenylindole dapi

Vaginal swabs were resuspended in phosphate buffered saline (PBS) and incubated on poly L-lysine coverslips (BD Biosciences, San Jose, CA) in a 24-well tissue culture plate for 30 min at 37°C. Coverslips were washed with PBS, fixed in 4% paraformaldehyde/PBS for 1 h, and permeabilized with 0.2% TritonX-100/PBS for 10 min. Fifty nanograms of fluorescently labeled probe targeting bacterial 16S rRNA was added to 200 µl of pre-warmed hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl (pH 7.2), 10% formamide, 0.01% SDS). Hybridization was carried out at 45°C for at least 2 h. Fluorescein-labeled broad range bacteria probe Eub338 (5′GCTGCCTCCCGTAGGAGT-3′) was used as a control. A Cy5-labeled specific probe (5′-TCCTCTTAGTGCCGTTCGTCC-3′) was used to detect “Ca. M. girerdii”. Following hybridization, coverslips were rinsed with pre-warmed hybridization wash buffer (0.45 M NaCl, 20 mM Tris-HCl (pH 7.2), 0.01% SDS) then incubated in the wash buffer for 15 min at 48°C. Immediately following, coverslips were washed with ice-cold distilled H₂O, dried for 15 min at 48°C and mounted with ProLong Gold Antifade containing 4′,6-Diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA). Slides were visualized by laser scanning confocal microscopy using a Zeiss LSM 700.

After dissection, individual midgut were deposited on slides, fixed in cold acetone for 15 min and rehydrated in PBS for 15 min. The midguts were then incubated for 2 h in Triton X-100 (0.2%). After washing with PBS, they were incubated for 30 min with PBS + 0.1% Tween 20 + 1% BSA. The slides were then incubated overnight at 4°C with anti-YFV-NS4B antibodies diluted 1:1000 in PBS. After washing with PBS, they were incubated for 1 h with secondary antibodies and washed with PBS. The actin network was visualized with phalloidin Alexafluor 488 (Invitrogen). After washing, nuclei were stained using Prolong gold antifade containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). All preparations were observed with a confocal microscope (ZEISS LSM 700 inverted) and images were acquired with the ZEN software.