Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron:dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe:dye was high), or both red and blue (when Fe:dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron:dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron:dye ratio is due to a simple carboxylate complex, [ Fe2H (dye)]-. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe2(dye)]2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.