Fitc conjugated goat anti rabbit igg

5 × 10⁴ of SSN-1 cells were seeded into a 24-well plate. After 24 h, cells were infected with wtRGNNV, rRGNNV, rRGNNV-B2-M1, or rRGNNV-B2-M2 at an MOI of 5 at 27 °C for 12 h and then replaced by media with L-15 medium containing 2% FBS for another 24 h. Then, infected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 15 min, and then perforated with 0.5% Triton X-100 in PBS for 15 min. Cells were blocked using 3% BSA in PBS for 30 min, and then incubated with rabbit anti-B2 polyclonal antibody at 27 °C for 1 h. After three times washing with PBS at room temperature, cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:500, Southern Biotech, Birmingham, AL, USA) at 27 °C for 1 h. Images of immunofluorescence were obtained by Olympus fluorescent microscope (Olympus Corporation, Tokyo, Japan). The percentage of B2 positive cells and mean fluorescent intensity of B2 positive cells were measured via Image J. Student’s t-test was used for comparison of the quantification data from three independent experiments. The p value < 0.05 was considered statistically significant (*).

Conjugation of 1–50 mer caveolin-1 peptide (PEPTRON, Daejeon, South Korea) to carboxyl beads (Bangs Laboratories, Fishers, IN, USA) using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) [27] (link). After washing the carboxyl beads with distilled water (DW), the carboxyl beads were incubated with caveolin-1 peptide 25 µg/ml (1–50) and 1% EDC (Pierce Biotechnology, Rockford, IL, USA) for overnight at 37°C with shaking. Then we add 10 mM of glycine and 10 mM glucose for 30 min at RT. Caveolin-1 peptide Conjugated beads were incubated with recombinant CSK or SHP-2 protein for 1 h at RT with shaking. After wash with PBS, beads were incubated with 1 µg of anti-CSK or anti-SHP-2 antibodies at 4°C for 1 h. The beads were then incubated with FITC-conjugated goat anti-rabbit IgG (Southern Biotech, Birmingham, AL, USA) or Alexa 488-conjugated goat anti-mouse IgG (Life Technologies, Grand Island, NY, USA) for 1 h and washed with PBS. Flow cytometric measurements were carried out on Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Experiments were repeated twice and showed similar results.