Fig. 2. Characterization of the MDC-labeled vacuoles. CHO cells were incubated in EBSS medium (starvation) at 37°C for two hours. Following this incubation period, cells were labeled with MDC as described in Fig. 1 and then incubated with 1 µM LysoTracker (LYS) for 10 seconds at RT. Cells were washed with PBS and immediately analyzed by fluorescence microscopy using the following filter system: excitation filter 510-560 nm, barrier filter 590 nm. MDC-labeled vacuoles are displayed in green and LYS in red. Arrows indicate some of the structures showing overlapping in the merged images (A-C). To label early endocytic compartments (EE), cells incubated under starvation conditions for two hours, were labeled with MDC and subsequently incubated with 0.5 mg/ml dextran tetramethylrhodamine for 5 minutes (D-F). To label late endosomes (LE), after the initial 5 minutes internalization of the endocytic marker, cells were washed and the probe was chased for 10 minutes before labeling with MDC. Cells were immediately analyzed by fluorescence microscopy (G-I). Endoplasmic reticulum (ER) was detected by indirect immunofluorescence (J-L). Cells were fixed, permeabilized and incubated with a rabbit antibody raised against ER membrane proteins (dilution 1:300) and with goat anti-rabbit Texas Red-conjugated secondary antibody. Cells were mounted with 50% glycerol in PBS and analyzed by fluorescence microscopy.