a) Endocytosis of fluorescently labeled dextrans in the salivary glands of live rats. Anesthetized rats were injected with Hoechst to label the nuclei (blue), and imaged in time-lapse by using two-photon microscopy. After 2:30 minutes, a 500 kDa FITC-dextran was injected to label the vasculature (green) and after 6:00 min a 70 kDa Texas-red dextran was injected to image the endocytic process. Endocytic structures appeared right after the injection and they increased in number and in size over time (see supplementary movie 4). Excitation wavelength 820 nm. Scale bar - 20 μm. b) Imaging lysosomal fusion in a live animal. Rats were injected with Alexa 488 dextran (green) and Mitotracker (red) and after 4 hours the submandibular glands were imaged in time-lapse by using single photon confocal microscopy. Two lysosomal structures were caught during a fusion event (inset). Note the dynamics of both the lysosomes and the mitochondria in supplementary movie 5. Scale bar- 5 μm. c-e Gene transduction in live animal. The acinar cells of the salivary glands of live rats were transduced by using plasmid DNA encoding for different genes as described in Sramkova et al., 2009. c) Cell expressing TGN38-mCherry, which show the typical TGN ribbon-like structure (red, arrows), and the water channel Aquaporin5-YFP (arrowheads), localized both at the apical plasma membrane and in vesicular structures (arrowheads). d) Cell expressing Life Act-GFP to label F-actin (Riedl et al., 2008). Note the enrichment of F-actin at the apical pole of the plasma membrane. e) Cell expressing LifeAct-GFP (green) and TGN-mCherry (red, arrow). Texas red dextran was also injected systemically in the rat and appeared in a blood vessel (arrowheads, supplementary movie 6). Scale bars - 5 μm.