CpG ODNs colocalize with endogenous SsTLR9 in primary salmon mononuclear phagocytes. A, The head kidney-derived cells were incubated on coverslips and following the indicated treatments the cells were processed for immunostaining and confocal microscopy as described in “Methods”. Treatment with fluorescent CpG-Cy5 (green pseudocolor) for 24 hours modestly up-regulated SsTLR9 which was detected in small granules. Pretreatment with IFN-γ for 48 hours enhances the SsTLR9 expression which upon addition of CpG-B is transported to vesicles that also contain CpG ODNs. Colocalization visible as yellow coloring in the merged images is indicated with arrows in the magnifications. The nuclei were stained with SYTOX Green (blue pseudocolor). NS stands for “no stimulation”. B, In live cells, CpG-B accumulates in tubular structures (arrowhead) and acidified vesicles (arrow) which, most likely, represent maturing endosomes. The cells were stimulated as described for panel A. Endolysosomes were stained with LysoSensor Green (pKa 5.2) and the cells were observed alive.