Oxidation of ruthenium red for use as an intercellular tracer

When ruthenium red (RR) is combined with OsO4, an electron-opaque complex forms which readily binds to the cell surface coat. However, the RR-OsO4 complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO4 containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO4 was allowed to stand 3 h before use, RR is oxidized by OsO4 to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO2=. Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO4 mixture resulted in a 50-80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO4 oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.