English: Confocal image stack of an isolated rat mesenteric collecting lymphatic labeled for ROCK1. Confocal slices were obtained in single photon mode at 2 μm intervals, which are indicated at the top left of each frame. Images of individual channels for ROCK1 (green) and nuclei, (white), and an overlay of these two channels are shown in the top row. Images of the glycocalyx (red) and smooth muscle (SM) actin (blue) channels, and an overlay are shown in the left column. All other combinations of overlays fill out the remaining rows and columns. In the bottom red/white overlay (bottom left), magenta areas indicate overlap. Just to the right, in the red/blue/white overlay, the endothelium and smooth muscle layers can be distinguished further due to the orientation of the nuclei. Endothelial nuclei are located in the inner layer and are oriented longitudinally. Smooth muscle nuclei are elongated and oriented perpendicular to the vessel, in the same fashion as the smooth muscle cells. In the green/red images, yellow pixels indicate overlap. In the green/blue images, cyan pixels indicate overlap. In the green/red/blue image, grey/white pixels indicate overlap. ROCK1 labeling overlaps with some areas of smooth muscle, to a lesser extent in the endothelial layer, and is also present within vasa vasorum on the outer surface of the lymphatic vessel. The labeling becomes weaker at the far end of the z-stack because no correction for z-distance was used in the image capturing. This vessel is representative of three separate experiments.