The interrelation of the size and substantivity of dyes; the role of van der Waals attractions and hydrophobic bonding in biological staining

Summary Using a pH signature criterion, it was found that whereas electrostatic attractions and repulsions were paramount in the binding of low molecular weight acid and basic dyes to tissue sections, high molecular weight dyes were also bound non-electrostatically.By studying the effects on staining of adding to aqueous dyebaths agents destroying the iceberg structure of water, the importance of hydrophobic bonding was established. It was noticed that the hydrophobic elastic fibres were stained by large dyes from dyebaths inhibitory both to electrostatic attractions and hydrophobic bonding (i.e. using acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths). This indicated that strong van der Waals attractions occurred, at least with some substrates. Supporting this idea was the observation that in tissue sections benzoylated before staining (i.e. made less acidophilic but more hydrophobic) additional structures were stained when using large acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths.Applications of the size-substantivity relationship were suggested, e.g. commenting on a standard stain for basic proteins; explaining the modes of action of traditional stains for elastic fibres and amyloid; rationalising the varied substantivities of tetrazolium salts; and finally suggesting guide lines for use in the design of new staining methods.     

The influence of inorganic salts when staining with preformed metal complex dyes

Summary The influence of various inorganic salts on the staining of tissue sections by a variety of preformed metal complex dyes (MCDs) has been studied. The staining patterns resulting were found to be extremely complex, though certain generalisations are possible.Usually the staining of basophilic tissue components (e. g. cartilage matrix and cell nuclei) was reduced as the amount of salt in the dyebath was increased. On the other hand the staining of some acidophilic tissue components (especially elastic fibres) was increased as the salt concentration rose. Both these effects can be rationalised by use of the Donnan equilibrium.The limited occurrence of salt-induced increases in staining intensity may be attributed in part to the extremely low rates of staining observed with acidophilic substrates.The decreases and increases in staining seen were to some extent dependant on the particular salt used, and presented an extremely complex picture. One general effect seen was that salts of Al³⁺, Cr³⁺, and Mg²⁺ had a greater inhibitory effect on staining than salts of K⁺ and Na⁺. This is explicable in terms of coordinative binding of the polyvalent ions by tissue substrates.On the basis of the information obtained comments were possible on various topics of biological staining interest. Thus it was concluded that of the MCDs studied only the aluminon-chromium complex showed evidence of binding by a mordanting mechanism. Further, it was found that the Critical Electrolyte Concentration theory was commonly inapplicable when staining tissue sections with MCDs.     

The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies

Synopsis An investigation of the role of phosphotungstic and phosphomolybdic acids in Mallory-like trichrome methods showed unexpectedly that, rather than acting as mordants to anionic dyes, these polyacids selectively blocked staining of all tissue components other than connective tissue fibres to Aniline Blue and other similar fibrereactive dyes. Connective tissue components were found to contain residues resembling histidine that are easily accessible to anionic dyes. Blocking towards typical anionic dyes for demonstrating plasma proteins, such as Biebrich Scarlet, was also demonstrated but was less complete. The blockade of both types of dye was labile if the staining times were extended; plasma dyes were more sensitive than fibre dyes in this respect. Histochemical reactions for tyrosine residues were blocked. In connective tissue, phosphotungstic acid did not block histidine residues demonstrable by the coupled tetrazonium reaction with previous iodination. Thus it is postulated that differential trichrome staining occurs by binding of Aniline Blue to basic residues in the connective tissue not blocked by phosphotungstic acid and subsequent replacement of the blocking agent by an anionic dye. The binding of phosphotungstic acid to both epithelium and connective tissue was demonstrated by the quenching of autofluorescence in these regions and by the reduction of the bound PTA to blue coloured products with titanium trichloride.     

A new principle in polychrome staining: a system of automated staining, complementary to hematoxylin and eosin, and usable as a research tool

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 millimicrons acrylic sections.     

Impurities and staining characteristics of Alcian Blue samples

Synopsis The composition of various samples of Alcian Blue^* and related dyes was studied, using t.l.c. (with cellulose as adsorbent andn-butanol: acetic acid: water as developing solvent), solvent extraction and precipitation, i.r. spectroscopy and classical semimicro analysis. All the Alcian Blue samples appeared to contain the same coloured components. The Alcian Green samples were mixtures of these blue components and Alcian Yellow. All the Astra Blue samples examined were composed of the same blue constituents. Colourless components identified were boricacid, dextrin and sulphate and sometimes amounted to nearly three-quarters by weight of the crude dyes. Impurities had only a slight effect on staining with Alcian Blue in aqueous acetic acid but appreciably affected staining by the critical electrolyte concentration (C.E.C.) procedure. Dextrin as impurity raised C.E.C. limits while the inorganic salt impurities raised the C.E.C. values of some substrates and lowered those of others.     

Mechanism of connective tissue techniques I. The effect of dye concentration and staining time on anionic dye procedures

Summary Anionic dye connective tissue procedures were performed by staining for 5 min and 24 h with (a) 0.00018m and 0.0018m solutions of 28 dyes, and 0.018m solutions of 21 dyes in saturated picric acid (SPA), and (b) 0.0018m and 0.018m solutions of 20 dyes in 1% (w/v) phosphomolybdic acid (PMA). The staining obtained with dyes in SPA was classified as selective (no cytoplasmic staining), moderately selective (traces of cytoplasmic staining) and non-selective (all other staining patterns). The staining of collagen and cytoplasm with dyes in PMA was separately classified on a scale of 1–5 (1 = no staining, 5 = maximum staining). The selectivity of the staining obtained with SPA with solutions of dyes at concentrations of 0.00018m and 0.0018m, and both staining times, was correlated (p < 0.001) with an empirical sulphonic acid constant (SAC) defined as the (number of dye sulphonic acid groups/dye molecular weight) × 10³. Correlation with molecular weight was poor and was significant only when staining was performed with 0.00018m dye solutions for 24 h. The dyes were divisible into three groups: group 1 (selectivity independent, or almost independent of staining time), group 2 (selective to moderately selective when staining was performed for 5 min), and group 3 (non-selective). The SAC of the group 1 dyes differed significantly from those of the group 2 and 3 dyes. Selectivity was essentially lost at dye concentrations of 0.018m. The staining with acidic dyes (no amines or substituted amines) in PMA differed significantly (p < 0.001) from that obtained with amphoteric dyes (containing basic substituents). In general, acidic dyes stained cytoplasm. Amphoteric dyes with the exception of indigocarmine stained collagen. However, most of these dyes also stained cytoplasm. In contrast to the results obtained with dyes in SPA, selectivity correlated strongly with molecular weight and only poorly with the SAC. Staining time and dye concentration affected selectivity only when the acidic dyes were used for 5 min at concentrations of 0.0018m and 0.018m. The data obtained do not permit a clear distinction between the rate control and chemical affinity models for the mechanism of staining with anionic dyes. However, it seems possible that different groups of dyes stain by different mechanisms. Part of this work was performed by M.I., S.N., M.J. and L.M. in partial fulfilment of the requirements for the completion of Pathology 438. A partial account of this work was presented at the annual convention of the British Columbia Society of Medical Technology, Victoria, British Columbia, October 1991.     

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions. Accepted: 3 January 2000     

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods

Summary The total protein staining of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye-protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen-Pararosaniline (SO₂) procedure for DNA, decreased Feulgen-DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.     

One-bath trichrome staining: Investigation of a general mechanism based on a structure-staining correlation analysis

Summary Dye pairs of contrasting colours were selected from acid dyes of varied chemical characteristics. The 44 dye pairs were investigated in a one-bath trichrome staining system in which the dye-baths were strongly acid. Dye concentrations, concentration ratios and staining times were varied for each dye pair. Thirty dye pairs stained collagen fibres distinctly different colours to muscle cytoplasm, while 14 dye pairs gave muddy, non-selective staining. Comparison of dye structures showed that in selective pairs the larger dye always stained the collagen fibres, with cytoplasm being coloured by the smaller species. With 28/30 of the selective dye pairs the differences in anionic weights of the dyes was > 200. However, in dye pairs giving non-selective staining, the anionic weights of the members of 13/14 of the dye pairs differed by < 200. As no other structural feature correlated so clearly with selectivity, it was concluded that the selectivity of one-bath trichromes is diffusion-rate controlled, involving the interaction of differentially permeable tissue sites (collagen being more permeable than muscle cytoplasm) with dyes diffusing at different rates (large dyes slower than small). In keeping with this, lengthening staining times reduced staining selectivity. The rate control mechanism suggested a rational trouble-shooting guide for one-bath trichromes, encompassing such practical factors as dye concentration, embedding medium, fixative, dye-bath pH, section thickness and staining time.     

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.     

Selective gray matter staining of human brain slices: optimized use of cadaver materials

We report a novel staining technique for human brain slices that distinguishes clearly gray from white matter. Previously described techniques using either Prussian blue (Berlin blue) or phthalocyanine dyes usually have included a hot phenol pretreatment to prevent white matter staining. The technique we describe here does not require hot phenol pretreatment and allows the use of brains stored for postmortem periods of one to two years prior to staining. Our technique involves staining with copper(II) phthalocyanine-tetrasulfonic acid tetrasodium salt 1% in water for 2 h followed by acetic acid treatment; this produces excellent blue staining of gray matter with little white matter staining. The stained brain slices are excellent for teaching human brain anatomy and/or pathology, or for research purposes.     

Mass transfer limit of fluorescent dyes during multicolor staining of aerobic granules

Multicolor fluorescence experiments are conducted to investigate the distributions of extracellular polymeric substances and/or cells in the bioaggregates. Successful staining requires that the dyes could fully stain the targeted substances of bioaggregates in a finite time. The mass transfer limit for one of the four fluorescent dyes, calcofluor white, concanavalin A conjugated with tetramethylrhodamine, Nile red, and SYTO 63, penetrating entire phenol-fed granules or those sectioned at 50 μm thick, was quantitatively determined. The former three dyes sufficiently stained the entire granule within prescribed time intervals. However, the SYTO 63 could not penetrate the 600-μm granule in a finite time. Simplified one-dimensional diffusional model estimated the apparent diffusivity of SYTO 63 in the aerobic granule matrix. This work revealed that each staining scheme should be examined for the possible mass transfer limit of dyes during staining.     

Van Gieson's picrofuchsin. The staining mechanisms for collagen and cytoplasm,and an examination of the dye diffusion rate model of differential staining

The staining mechanism of van Gieson's picrofuchsin was studied by use of simple protein model systems and tissue sections, and by spectrophotometry and dialysis experiments. At the endpoint of the staining reaction (equilibrium) cytoplasm is yellow. Dye dilution experiments demonstrated that the highest affinity in the tissue section — picrofuchsin system is between binding sites in cytoplasmic protein and acid fuchsin. Nevertheless sections that were first stained in acid fuchsin (AcF) and then in picrofuchsin ended up with cytoplasm stained yellow. It was concluded that differences in the dye diffusion rates and differences in the permeability of tissue components cannot be invoked to explain the differential staining result. Model experiments with dissolved proteins demonstrated a positive relationship between protein concentration and uptake of picric acid (PA) from picrofuchsin. From this and experiments with additives (sodium dodecylsulphate, urea etc.) and organic solvents, it is proposed that coagulant interchain cross-linking at the high protein concentration of the cytoplasm masks potential dye-binding sites. This affects high affinity dyes with multiple binding sites more than small dyes, and so puts AcF at a disadvantage compared to PA. Staining of non-collagen proteins is mainly by hydrophobic bonding, involving ionic attractions, apolar bonds, and release of water. This mode of binding is relatively strong, decreases swelling and leads to slow dye exchange. Dye binding to collagen is mostly by hydrogen bonds, but in aqueous dye solvent nonpolar residues and charged residues may also participate. This structure remains relatively open during and after dye-binding, and the bound dye ions are therefore easily exchanged for other dye ions. Address at which the main part of the investigation was carried out     

Investigation of staining,polarization and fluorescence microscopic properties of myoepithelial cells

Summary During studies of early arteriosclerotic lesions fibers with the staining properties of myosins were observed in epithelial cells of various organs. To obtain a basis for further studies, staining, oolarization and fluorescence microscopic properties of classical myoepithelial cells and tonofibrils were investigated. The tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN stain for myosins and related proteins was applied to sections of tongue and skin. In other series various milling dyes, xanthene dyes and Thiazine Red R were substituted for Levanol Fast Cyanine 5RN.Myoepithelial cells of lingual and eccrine sweat glands showed the microscopic properties of smooth muscle cells; tonofibrils had little or no affinity for the dyes tested. The terminal bar-terminal web system of glandular epithelium and the fibrous layer in ducts of eccrine sweat glands resembled myosins and differed significantly from proteins of the epiderminkeratin group, e.g. tonofibrils. In preliminary studies the iodinated xanthene dyes Rose Bengal G, Erythrosin B and Y were found suitable for light, fluorescence and electron microscopic studies.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.     

A convenient buffer system for controlled staining with basic dyes

Summary A new buffer system is described for use in histochemical staining with basic dyes. The buffer is made up by mixing solutions of formic acid and sodium acetate. Tables giving the proportions for closely-spaced pH values in the range 3.0–5.6 are presented. A table for an acid phosphate series down to a pH of 2.0 is also included. The value of these buffer mixtures in histological as well as histochemical staining with basic dyes is stressed.With 1 Figure in the Text     

The accentuators,pH and the principles of the effect in histological staining

A study was made of the influence of accentuators (phenol, aniline) and pH on the stainability of vegetable tissue (roots ofAllium cepa L.) with light green and methylene blue with reference to the theory of the electrostatic basis of staining. — The results obtained on the whole confirmPischinger’s theory of the electrostatic basis of staining for light green only, but with methylene blue the situation was found to be much more complicated. Only the alkalization of a watery solution of methylene blue increases its staining ability, whereas the accentuators markedly disturb staining. Prischinge’s theory is thus not of general validity, but applies only to one factor of this complicated process. Electrostatic influences acting on staining ability are not so important as other factors in this complicated process in some dyes.     

A New Principle in Polychrome Staining: A System of Automated Staining, Complementary to Hematoxylin and Eosin, and Usable as a Research Tool

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 mμ acrylic sections.     

Some observations on the mechanisms of blocking of nuclear staining by cisplatin

Summary The effect of cisplatin (cis-dichloro-diamminoplatinum II) treatment on staining of nuclei with various basic dyes and with the Feulgen reaction has been studied. Although cisplatin is reported to show negligible reaction with DNA phosphates, it has a substantial blocking effect on staining with most dyes. Short treatment with cisplatin results in binding mainly to guanine bases of DNA, causing partial blocking of the Feulgen reaction and almost complete blocking of ethidium intercalation; binding of neutral red and crystal violet is enhanced, apparently as a result of cisplatin-induced denaturation of DNA. Very prolonged cisplatin treatment does not completely block the Feulgen reaction, indicating that reaction of cisplatin with purine bases is not complete. Since attachment of cisplatin to DNA bases is unlikely to prevent binding of most basic dyes, it is suggested that the blocking of their staining may result from steric hindrance caused by formation of DNA-protein cross-links by cisplatin. Whatever the mechanism, it is incapable of producing complete blocking of staining with certain dyes. As a practical tool, it appears that rapid and almost complete blocking of staining by cisplatin may be used as an indicator of intercalative binding of dyes to DNA.     

A new version of the Ag?NOR technique. A combination with DAPI staining

Summary The Ag−NOR staining technique is widely used for visualizing nucleolar organizer regions (NORs) in various plant and animal tissues. We describe a simple and time-saving combination of Ag−NOR staining with DNA detection by fluorescence microscopy. This modification was tested on cultured cells and semi-thin sections of plastic-embedded tissues. Of the different fixatives and embedding media used in our studies, the best results (i.e., high selectivity of staining, and lack of or very low background precipitation) were obtained with fixation in methanol-acetone at −20°C for cultured cells, and fixation in 4% formaldehyde followed by embedding in Histocryl resin for tissue sections. The optimal time of Ag−NOR staining was determind experimentally for all materials tested. The specificity of the staining was checked at the electron microscopical level. Especially good results were obtained by mixing epifluorescence with standard bright-field illumination. In such a combination, Ag−NOR-positive nucleoli, or their fibrillar centres and dense fibrillar components, were clearly visible against a bright background of nuclear DNA.