Ruthenium red and violet. II. Fine structural localization in animal tissues

Abstract

The inorganic dye, ruthenium red, stains extracellular materials in animal tissues which probably are acidic mucopolysaccharides. It complements other techniques, its advantages being fine grain, high resolution and good contrast. Localization is shown in mouse and rat muscle, heart, lung and intestine, frog cartilage and cells scraped from oral epithelium of human beings. Attention is paid to collagen bundles, the cell/collagen interface and particularly the myotendinal junction, cartilage matrix and agar gel, desmosomes, intestinal microvilli, erythrocytes and vascular endothelium, nerve fibers and the T-system of striated muscle. Although ruthenium red generally is excluded by plasma membranes, it penetrates giving intracellular density, if the membrane is broken. Even when the cell membrane is intact, exceptions occur with selective staining of the T-tubules or the sarcoplasmic sacs depending upon the state of contraction of the muscle cell, and with intracellular staining of certain nuclei and epithelial cells. Ruthenium red stains intracellular lipid droplets revealing lamelJae, and stains myelin forms grown from crude egg lecithin but cannot penetrate deeply. It is localized in extracellular materials which have an important mechanical function. Its exclusion by cell membranes permits tracing tortuous cellular invaginations and those exceptions to its exclusion invite a comparison of the localization of the dye with the function of the cell.Cells advertise themselves to the outside world and to each other by their surfaces. Scientists, however, are better acquainted with cells in terms of their internal organelles and molecules, having violated their privacy with microscopes, homogenizers, and separation procedures. The research directed toward this outer surface, called the plasma membrane, has been concerned mainly with the lipids and proteins which are known to reside there. However, during the 1960's evidence accumulated prompting consideration of another component which is minor in terms of dry mass but major in terms of function. This third component is polysaccharide, usually present as a glycoprotein. Its importance was strongly emphasized from a morphological standpoint, perhaps f m t by Bennett, who, in 1963, coined the general term "glycocalyx," although Chambers by 1940 had designated similar layers in other cells as "extraneous coats," and their history can ANAT. REC., 171: 369-416.be traced much earlier. In 1966, Rambourg, Neutra and Leblond published an important paper after examining a wide variety of tissues with several histochemical tests for polysaccharides. They concluded that "nearly all cells investigated are covered by a thin but definite band of stained material indicating the existence of a surface layer." They also felt that "the layer is not part of the plasma membrane itself but is a surface coat." The biochemical difficulties which hindered work on these substances as well as their general importance in biology has been elegantly reviewed recently by G. M. W. Cook ('6...

Proteoglycans in primate arteries. I. Ultrastructural localization and distribution in the intima.

“…They are approximately the same size as cartilage matrix granules and possess similar 30-60-A thick filamentous projections which appear to connect adjacent granules. Furthermore, they are deposited on the major period band of collagen fibrils in a pattern similar to that observed in cartilage (1,9,26,31,39,41,46). These results indicate that arterial collagen possesses similar proteoglycan binding sites at their major period bands to those suggested for cartilage collagen (39,41).…”

“…The distribution of ruthenium red-positive matrix granules in normal and hyperplastic intimas is remarkably similar to the distribution of similarly stained granules in cartilage matrix (9,24,26,46). They are approximately the same size as cartilage matrix granules and possess similar 30-60-A thick filamentous projections which appear to connect adjacent granules.…”

“…Ruthenium red, a hexavalent cationic dye, has been shown to bind to proteoglycans, among other substances (24,26) and, if added at the time of fixation, appears to facilitate the preservation of these macromolecules by precipitating them and rendering them insoluble.…”

“…Glycosaminoseveral connective tissues (1,4,9,18,23,26,28,31,33,38,41,42,46,47), relatively little is known concerning the ultrastructural distribution of these macromolecules in arteries.…”

Immunological escape mechanism in spontaneously metastasizing mammary tumors.

“…The glycocalyx of the tumor cells was demonstrated by staining this structure with ruthenium red by the method of Luft (2,3), with a Siemens Elmiskop IA. Ruthenium red is an inorganic dye said to bind specifically with the acid mucopolysaccharides of the cell surface coat, bringing out the glycocalyx as an intensely electron dense layer over the luminal surface of glandular epithelial cells by catalyzing the precipitation of osmium in the fixative.…”