2.7. Immunofluorescence Assay to Detect TiLV in the RHTiB Cell Line

2.7. Immunofluorescence Assay to Detect TiLV in the RHTiB Cell Line

An immunofluorescence assay (IFA) targeting TiLV was used to confirm the inhibition of viral entry into RHTiB cells (a cell line from the brain tissue of red hybrid tilapia) [48]. Briefly, the RHTiB cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B at a pH of 7.4 at 25 °C without CO₂. When the cells achieved 70% confluence, they were trypsinized, counted, and diluted to a concentration of 2 × 10⁵ cells/mL. Subsequently, 500 µL of the cell suspension was seeded onto cell culture slides (SPL Life Science, Pocheon-si, Gyeonggi-do, Republic of Korea) in an L-15 medium containing 10% FBS and incubated until 80–90% confluence was achieved. The cells were infected using different methods: 100 µL TiLV mixed with 100 µL L-15 medium (positive control), TiLV mixed with a 1:2 or 1:10 IgY solution, TiLV mixed with IgY at T0, control egg, and control L-15 medium (negative control). These mixtures were incubated at 25 °C with continuous shaking at 400 rpm for 2 h (Eppendorf^® ThermoMixer^® C, AG, Darmstadt, Germany). The cells were washed twice with an L-15 medium without FBS, incubated with the virus mixtures for 1 h, and subsequently replaced with an L-15 medium containing 2% FBS, followed by incubation at 25 °C for 24 h.

The cells were fixed with ice-cold 100% methanol for 10 min and washed twice with PBS. They were then treated with 0.3% Triton X-100 in PBS for 10 min and washed again with PBS. The membrane was blocked with 2% BSA in PBS for 30 min to prevent nonspecific binding, followed by overnight incubation at 4 °C with the primary antibody (IgG of TiLV) in a blocking solution at a 1:100 dilution. The cells were then incubated with the secondary antibody (goat anti-rabbit IgG H&L Alexa Fluor^TM 488; Abcam, Carlsbad, CA, USA) in PBS at a dilution of 1:500 for 1 h at room temperature and washed with PBS. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) at a 1:1000 dilution for 15 min, washed with PBS, mounted with ProLong^TM Gold Antifade reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on glass slides, and then a cover glass was placed over them. All images were captured using a confocal microscope (Fluoview 3000, Olympus, Tokyo, Japan), which confirmed the specific binding of IgY to the TiLV-infected cells through the colocalization of the DAPI and Alexa Fluor signals. The fluorescence intensity values were quantified using cellSens Dimension software version 2.3 (Olympus, Tokyo, Japan). Five areas were randomly selected, and the fluorescence intensity of the green signal, which indicated positive TiLV-infected cells, was measured. The green signal intensity was analyzed as mean ± standard deviation (SD) and compared with the positive control, egg control, and TiLV mixed with the 1:2 or 1:10 IgY solutions.