Ultrastructural studies of bovine retinal microvascular basement membranes with the cationic dye ruthenium red

Our recent observation that the basement membranes of brain microvessels do not stain with the cationic dye ruthenium red has raised the question of whether the basement membranes of this and other vascular beds functioning as barriers between blood and neural tissues are deficient in the polyanionic macromolecules, such as glycosaminoglycans, which are responsible for the ruthenium red staining of other vascular basement membranes. We therefore attempted to produce staining in the only barrier-type microvascular basement membrane known to contain heparan sulfate. Bovine retinas were fixed by immersion in aldehyde fixatives containing ruthenium red, buffered with either 10 mM or 100 mM sodium cacodylate. We found discrete, electron-dense deposits of ruthenium red in vascular basement membranes, quite similar to those seen in vascular basement membranes of nonneural tissues after exposure to ruthenium red. These deposits were more distinct and more frequent in tissue exposed to ruthenium red-aldehyde solutions buffered with 10 mM cacodylate. They were not seen if ruthenium red was omitted from the fixative. The results demonstrate that anionic macromolecules in basement membranes of barrier-type microvessels can be stained with cationic dyes, and suggest that the failure of brain microvessels to stain with ruthenium red may be the result of a relative or total lack of polyanion in this basement membrane, or of other unique properties.