Rabbit anti mouse igg fitc

Flow cytometry analysis was performed to characterize the cell surface antigen of the fibroblasts. FACSCalibur (Beckton-Dickinson, San Jose, CA) was used and a minimum of 10,000 events counted with Cell Quest software (Beckton-Dickinson Labware, Franklin Lakes, NF). Cells were trypsinized, centrifuged, and incubated with primary antibody for 15 minutes on ice in a dark condition. They were then washed with PBS containing 3% fetal bovine serum (FBS; Sigma) and 0.05% Na₂N₃, and then were incubated with rabbit anti-mouse IgG/FITC (Dako) as a secondary antibody for 15 minutes on ice in a dark condition. Washing with PBS again, a FACS scan was performed using Cell Quest software. The primary antibodies used are described in S2 Table.

Vero cells were separately transfected with individual recombinant plasmid constructs (pCMVkanD1prME-pCMVkanD4prME) using lipofectamine 2000 (Invitrogen). At 24 hr post-transfection, cells were fixed, permeabilized and stained with flavivirus-reactive anti-E antibody (clone 4G2) [22] (link) and anti-DENV-NS1 antibody (clone DN3, Abcam). Rabbit-anti-mouse IgG-FITC (Dako) and goat-anti-mouse IgG-Alexa-fluor (Molecular Probe) were used as secondary Ab for detection of anti-E and anti-NS1, respectively. Cell nuclei were counter stained with 4, 6-diamino-2-phenylindole hydrochloride (DAPI) (Sigma–Aldrich). Stained cells were visualized under fluorescence microscope. Western blot was used for detection of E protein expression in cells culture supernatant at 24 hr post-transfection or infection by using 4G2 mAb. The cell culture supernatants (crude) were directly subjected for protein detection, transfected cells were not lysed before supernatant collection. Rabbit-anti-mouse IgG conjugated with horseradish peroxidase (KPL) was used as secondary Ab and detected by chemiluminescence substrate (Immobilon western, Millipore) then exposed to an X-ray film. Vero cells infected with DENV-2 (strain 16681) at the multiplicity of infection of 0.5 or transfected with empty pCMVkan expression vector were employed as positive and negative controls, respectively.