Ruthenium Red—p-Phenylenediamine Staining of Monolayers to Facilitate Handling and Selection of Specific Cells for Transmission Electron Microscopy

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Abstract
The handling of monolayers for transmission electron microscopy has presented many problems, the main one being difficulty in visualizing the monolayers after polymerization of their plastic embedment following conventional glutaraldehyde-osmium fixation. The application of ruthenium red—p-phenylenediamine during processing produced intensely darkened cells which could be examined and photographed either in 95% ethanol or following Spurr embedment without further treatment or sectioning. This treatment also facilitated orientation of the monolayers when re-embedding, and permitted precise localization of monolayers within flat embedding molds when trimming and thin sectioning for transmission electron microscopy. Increased color density is the combined result of more complete retention of soluble elements during initial fixation by ruthenium red and the formation of a colored reaction product between the bound ruthenium red and osmium which is further intensified by p-phenylenediamine.