Scanning electron microscopy of the ependymal surface of the third ventricle after silver nitrate staining

Abstract

The suitability of silver nitrate as a stain for light microscopic (LM) and scannning electron microscopic (SEM) study of ventricular ependymal cell boundaries was investigated. Wholemount preparations of rabbit third ventricle viewed ‘en face’ with the LM, reveal a distinct and uniform network of intercellular silver lines outlining the borders of polygonal ependymal cells that vary greatly in size and shape. These cells do not exhibit any specific orientation except in certain circumscribed regions of the ventricle. The same results were obtained whether the ventricular ependyma was formalin fixed after staining or formaldehyde-glutaraldehyde fixed before. Viewed with the SEM, the ependymal cells showed the same distinct outlines. The interependymal boundaries are marked by elevated silver deposits that appear brighter than the sorrounding tissue. Of the two preparative procedures investigated for SEM, in situ fixation before silver staining provided the best preservation of fine surface structure. However, prominent intercellular silver deposits were also obtained when fixation by immersion followed staining. Subsequent post-fixation in OsO 4 and/or dehydration in graded alcohol erodes intercellular silver deposits, such that they cannot be resolved with the SEM. On the other hand, rapid alcohol or acetone dehydration provides optimal preservation of structural detail, while also permitting satisfactory resolution of interependymal silver deposits. There are, however, subtle differences both in the appearance of the ependymal surface and the silver outlines depending upon the dehydrant used. The use of silver nitrate in connection with LM or SEM of ventricular ependyma nervertheless permits convenient identification of individual cells and their differentiation from other structures.