Figure 3. Trafficking of dextran from the early endosomes to the late/endosomes lysosomes.

Rats were injected with TXR-D and after 24 hours either euthanized (A) or injected with 488-D (B-E). A – SSGs were isolated, fixed and labeled with an antibody directed against Lamp1 (green) and Hoechst 33342 (blue) Images were acquired by confocal microscopy. TXR-D is localized in structures decorated by Lamp1 (insets) both in cell located at the surface (left panels) and deeper in the tissue (right panels). Bars 20 µm. B – Twenty, and 30 minutes after the injection of the 488-D, the rats were euthanized, the glands were excised and imaged. At 30 minutes but not at 20 minutes a significant co-localization between 488-D and TXR-D was observed (insets, arrowheads). Bars 10 µm. C-E – Dynamic imaging of the trafficking of dextran through the endosomal compartments of live animals. - Before the injection of 488-D the glands were exposed and imaged at 830 nm. TXR-D containing lysosomes are shown in red - C – In the first 25 minutes after the internalization, endosomes grow in size but do not fuse with lysosomes. Bars 20 µm. D - Small early endosomes undergo fusion (arrowheads). Bars 20 µm. E – After 55 min from the injection we observed a 488-D containing endosomes (time 55:00, inset arrowhead), which acquired first TXR-D (time 56:38, inset and arrowhead) and then fused with a structure containing both 488-D and TXRD (time 57:57 and 58:14, insets and arrows). Bars 10 µm.