(A) PBMC of four HAM/TSP patients (PH1485, PH1593, PH1601, and PH1624) were stained with the 4D4-F3 anti-HBZ mAb followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and (B) with the A51-2 anti-Tax-1 mAb followed by Alexa Fluor 488-conjugated goat-anti-mouse IgG2a antibody (green), and analyzed by confocal microscopy. Specific counterstaining of nucleus or cytoplasmic compartments was performed by using DRAQ5 fluorescence probe to detect the nucleus and anti-vimentin rabbit polyclonal antibody followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488 (green, panel A) or to Alexa Fluor 546 (red, panel B) to stain the cytoplasmic compartment. At least 300 cells were analyzed; a representative cell for HBZ or for Tax-1 staining is shown for each patient.(C) Low magnification field to show the co-existence of cells mutually exclusive for the expression of cytoplasmic HBZ and Tax-1 in PBMC of patient PH1624. Cells were co-stained with 4D4-F3 anti-HBZ mAb and with A51-2 anti-Tax-1 mAb followed by specific secondary antibodies staining as described in (A) and (B).