Immunofluorescence analysis of S2/ZF-ZR and S2/JEF-JER cells with antibody 2F2. S2 cells were transfected with pAc/ZF-ZR (frame A) or pAc/JEF-JER (frame B). Transfected cells were harvested in PBS and plated onto microscopic slides. After air drying, the cells were fixed in 10% formalin with 1% Triton X-100 at room temperature for 30 min. The cells were incubated subsequently with JEV E specific monoclonal antibody 2F2 and a fluorescein-conjugated secondary antibody (DAKO) at room temperature for 1 hr. Each incubation was followed by two washes in PBS. Finally the slides were mounted with VECTASHIELD Mounting Medium containing propidium iodide (Vector Laboratories) and viewed under an Olympus fluorescence microscope. The green fluorescence indicates the detection of JEV E proteins.