Identification of HTLV-1-PR:hFcγ1 Recombinant Protein by the Co-localization Assay

The cells containing the stable expression of HTLV-1-PR:hFcγ1 fusion protein were incubated with anti-Fcγ1 human antibody conjugated with fluorescein isothiocyanate (FITC; BioLegend, USA) to visualise secreted recombinant fusion protein in the sample via a fluorescence microscope (Nikon Eclipse E200, Japan).

Cell suspension of (4 × 10⁶, 2 μL) were placed on immunofluorescence slides, dried overnight at 4 °C, and then fixed with methanol/acetone at − 20 °C. Cells were permeabilised with 0.2% (v/v) Triton-X 100 in PBS for 30 min and blocking was performed for 15 min with 3% (w/v) BSA (Sigma, USA) in PBS. The primary antibody was added at the optimal concentration, diluted in 3% (w/v) BSA in PBS, and incubated in a humidified chamber at 37 °C for 90 min. Next, slides were washed with 0.1% (w/v) BSA in PBS, and the secondary antibody was added at the optimal concentration of 3% (w/v) BSA and incubated in a humidified chamber for 60 min. Cells were washed in PBS in 0.1% (w/v) BSA and stained with ethidium bromide. After fixing the coverslip with a fluorescent mounting medium (Citifluor, UK) and sealing with nail varnish, samples were viewed by a fluorescence microscope.