Fig. 6. Endosomes of the transcytotic pathway remain distinct in BFA-treated cells. 3-D stereopair images of living, polarized PTR cells were collected in the presence of basolateral Tf conjugated to both fluorescein and rhodamine (FR-Tf). The pH-sensitive fluorescence of fluorescein gives this probe a green fluorescence in the neutral environment of probe bound to the plasma membrane (note bases of cells in A), but an orange fluorescence for Tf in acidic endosomes. Whereas Tf is restricted to acidic endosomes in control cells (A), it is found in both acidic medial endosomes and relatively alkaline apical compartments in cells previously incubated with BFA for 15 minutes, and then in BFA with FR-Tf for another 22 minutes (B). Panels A and B are 34 µm in height.