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High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Figure 8

HIM imaging of external gold labeling in the kidney.

(A) Lower magnification of a modified PLP-fixed proximal tubule with its brush border after labeling of surface glycoproteins (and/or glycolipids) with gold-conjugated WGA. The tissue was dehydrated using the rapid graded methanol procedure. The gold particles appear as discrete, white globular entities associated with the external surface of brush border microvilli and other parts of the cell surface adjacent to the microvilli. Bar = 1 µm. The gold label can be seen more easily at higher magnification (B - arrows), where it extends along the entire length of the microvilli. Bar = 200 nm. The inset in panel B shows a modified PLP-fixed proximal tubule brush border that has been immunolabeled with a monoclonal anti-megalin antibody followed by a secondary, gold-conjugated anti-mouse antibody. In this case, the pale gold particles (arrows) are concentrated towards the base of the microvilli and do not extend along their entire length (inset; Bar = 200 nm). (C) The apical surface of a collecting duct principal cell from the same kidney immunolabeled with the anti-megalin antibody and the respective gold-conjugated secondary antibody. The image shows no gold particles, attesting to the specificity of the proximal tubule megalin binding. Bar = 500 nm.

Figure 8