Microanatomy of human ASL. (a) WD human airway epithelial culture exhibiting rotational mucus transport (see Figure 3), fixed with perfluorocarbon/osmium. Note a distinct mucus layer atop a distinct PCL. (b) Visualization of glycocalyx on WD human airway epithelia by the freeze substitution technique. Note the high degree of organization of this barrier. (c) Left: X-Z confocal image of living WD human airway epithelial culture. The cells were stained with calcein, AM, (green), and the ASL was visualized with Texas red dextran. Scale bar = 10 μm. Top right: Fluorescent “dissection” of mucus layer and PCL in living WD airway epithelia by confocal microscopy. The mucus layer is visualized as green fluorescent beads and the PCL as the “bead-free zone” interposed between the mucus layer and cell surface (black). Scale bar = 10 μm. Bottom right: Detection of glycocalyx by fluorescence/confocal microscopy. The keratan sulfate component of the glycocalyx is visualized by Texas red–labeled anti–keratan sulfate (anti-KS) antibodies. Scale bar = 5 μm.