A combined perfusion- and immersion prefixation with glutaraldehyde followed by a tannic acid/arginine/osmium tetroxide (TAO) treatment of the guinea pig cochlea is described for field-emission gun scanning electron microscopy (FEG-SEM) observation of the fine structure of the stereocilia of the organ of Corti. Conventional osmium tetroxide postfixation methods in combination with a thin conductive coating failed to show the fine structure of the glycocalyx of the epithelial lining in the endolymphatic compartment of the cochlea, in particular, on the stereocilia surface. The antennulae-like glycocalyx covering of the stereocilia surface of the more pronounced rows of outer hair cells has been demonstrated only in ultrathin sections by means of cationic markers. The side- and tip-links connecting the stereocilia have been demonstrated both in scanning and transmission electron microscopy, although at that time these structures often were considered as artificial. However, they can be visualized with FEG-SEM at low accelerating voltage (2-3 kV), and at appropriate working distance and probe current, in combination with a glutaraldehyde perfusion/immersion prefixation and TAO postfixation. Stereo images enhance considerably the three-dimensional appreciation of the stereocilia with glycocalyx lining and side- and tip-links, proving that these connections are a structural part of the hair cell.
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