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

Abstract

K E Y w ORDS. Chromoxane cyanine R, eriochrome cyanine R, histological staining, myelin staining, nuclear staining, solochrome cyanine R, staining mechanisms, staining methods, mordant dyes, differentiation of stains. S U M M A R YThe 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(II1) 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, [FezH(dye)]-. The blue colour would then result from withdrawal of a proton from the red complex to give [Fez(dye)I2-. 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. I N T R O D U C T I O N Some dyes can be used in microtechnique for several different purposes. The most important example is haematein, a hydroxyketone formed by oxidation of haematoxylin. Haematein is used in conjunction with a metal salt as mordant. The different staining effects result from variation of the metal, the composition of the solutions, the times of exposure and the conditions of differentiation. Synthetic dyes can be used in some of the applications of haematoxylin 8 1984 The Royal Microscopical Society 25

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

“…Further modifi cation of the technique of Hyman and Poulding (1961) was introduced by Kiernan (1984b). Although using a working solution with relatively low iron:ECR ratio, he emphasized that to obtain differential dichromatic staining that resembled standard H & E, the working solution needed to be at pH 1.5.…”

“…Although using a working solution with relatively low iron:ECR ratio, he emphasized that to obtain differential dichromatic staining that resembled standard H & E, the working solution needed to be at pH 1.5. Kiernan (1984b) recommended further that after a few minutes in the working solution, sections should be washed in distilled water, then dehydrated in ethanol.…”

“…Although he did not use a mordant, he obtained blue stained nuclei, and red staining of most other tissue components. Both Clark (1979) and Kiernan (1984b) suggested that iron in the tap water or the dyestuff itself had produced the dichromatic staining. Hyman and Poulding (1961) added iron alum intentionally to the acidifi ed aqueous solution of ECR and obtained dichromatic staining, which they recommended for fresh or formalin fi xed frozen sections.…”

“…Careful differentiation after staining with Fe-ECR permits both blue and red complexes to stain basophilic or acidophilic tissue components, respectively. The several methods in the literature that describe dichromatic staining with Fe-ECR (Pearse 1957, Hyman and Poulding 1961, Chapman, 1977, Kiernan 1984b ostensibly produce staining similar to hematoxylin and eosin (H & E). I describe here a new Fe-ECR dichromatic procedure.…”

A system for quantitative evaluation of fixatives for light microscopy using paraffin sections of kidney and brain

“…Tissues darkened by the osmium tetroxide in Altmann's fixative were bleached by treating the hydrated sections with 0.5% potassium permanganate followed by 5% sodium thiosulfate. The sections were stained with iron alum-eriochrome cyanine R using a modification (Kiernan 1984(Kiernan , 2008 of the method of Hyman and Poulding (1961). This one-step technique provides results similar to those obtained with hemalum and eosin.…”