In order to study all stages of DVs in the same cell we used a protocol of sequentially exposing cells to different sizes of latex beads interspersed with washes: for example, 3 minutes in 0.1µm beads, wash 3 min, 3 minutes in 0.3µm beads, wash 3 min, 3 minutes in 0.6µm beads, wash 3 min, 3 minutes in 0.8µm beads, wash 3 min and finally 3 minutes in 1.1µm beads followed by fixing the cells. Thus the 1.1, 0.8, 0.6, 0.3 and 0.1µm bead-containing vacuoles will be 0-3 min, 6-9 min, 12-15 min, 18-21 min and 24-27 minutes old, respectively. The electron micrograph shown here was of a DV-II that was surrounded with docked secondary lysosomes. Secondary lysosomes only recognize and dock at the DV-II membrane. They do not dock with DV-I or DV-III vacuoles. TEM taken on 2/27/81 by R. Allen with Hitachi HU11A operating at 75kV. Neg. 7,000X. Bar = 0.5µm.
Standard glutaraldehyde fixation followed by osmium tetroxide, dehydrated in alcohol and embedded in an epoxy resin. Microtome sections prepared at approximately 75nm thickness. The negative was printed to paper and the image was scanned to Photoshop. This digitized image is available for qualitative analysis. Additional information available at (http://www5.pbrc.hawaii.edu/allen/).
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