Ruthenium Red Staining of Chromatin in Epon Sections

For a long time, the ammoniated ruthenium oxychloride “ruthenium red” (today formulated [1, 2] as [(NH3)5 R u •O • Ru (NH3)4 O • Ru (NH3)5]6+ • Clg), has been used as a microscopic test for pectins [2, 3]. Several applications of this contrasting agent for electron microscopic detection of acid polysaccharides and proteoglycans have been stud­ ied [4-10]. Its use as a Schiff-like reagent was reviewed recently [11], However, it seems to have been overlooked that ruthenium red (RR) could also stain nuclei [12], and only scarce observations on the contrast enhancement of chromatin and DNA by RR have been reported [4, 11, 13]. The strong binding of RR to polyanionic macro­ molecules [10-13] brought to our attention the pos­ sibility that it could serve as a staining and con­ trasting agent for chromatin in Epon sections. A drawback of this approach, however, is the poor penetrability of plastic-embedded tissues by aqueous solutions of RR [4], In order to overcome this dif­ ficulty, staining of semithin sections was attempted under several different conditions (7% acetic acid, 50% ethanol, increasing RR concentrations or stain­ ing time, heating, etc.). Among these, acceptable results were only achieved by using RR in 50% ethanol. A fresh 0.5% solution of RR (Scharlau) in distilled water was diluted 1:1 (v/v) with absolute ethanol. After 24 h a dark brown, flocculent preci­ pitate is separated and the remaining bright red solution can be used after filtration or centrifugation.