Staining of macromolecules: possible mechanisms and examples.

This review is based on a presentation given at the Biological Stain Commission meeting in June 2008. I discuss staining as an interaction between dye, solvent, and biological macromolecules. Most staining takes place in water, where the physico-chemical properties of the macromolecules are particularly important. Staining from aqueous solution is summarized. The first step is diffusion-ion exchange, which builds up the dye ion concentration close to the appropriately charged tissue constituents. While charge interactions are important for selectivity and build-up of dye ions around specific tissue and cell constituents, they have in most cases little to do with actual dye binding. The next step, actual binding, is predominantly between aromatic and other non-polar parts of the dye and corresponding groups in the tissue constituent. This results in a reduction of the total hydrophobic area exposed to water, hence the term hydrophobic interaction. Because dye binding is predominantly by dispersive forces, the larger the aromatic dye system and the fewer the number of charges on the dye, the greater the substantivity or affinity. Some relatively straightforward anionic or cationic one-step staining systems are discussed also. These include amyloid staining with Congo red, elastin staining with orceins, collagen staining with picrofuchsin, DNA-RNA staining with methyl green-pyronin Y, acid heteroglycan staining with Alcian blue, and metachromatic staining.

The role of glycine and prolines in connective tissue fiber staining with hydrogen bonding dyes.

Extensive hydrogen bonding of dyes to connective tissue fibers is made possible by the high content of the amino acids proline and glycine in elastin and collagens. Proline confers an extended polypeptide structure and glycine is the only amino acid whose specific side group, -H, is so small that it forms no obstacle to hydrogen bonding between the peptide group and external molecules. Thus, a high proportion of the peptide groups in fibrous proteins are directly accessible to hydrogen bonding groups dye molecules.

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates.

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.

Standardized staining methods: Feulgen-Rossenbeck reaction for desoxyribonucleic acid and periodic acid-Schiff (PAS) procedure.

A project group working under the European Confederation of Laboratory Medicine (ECLM) presents recommendations for standardized procedures for the Feulgen-Rossenbeck-Schiff and the periodic acid-Schiff (PAS) reactions on cytological and histological material. The advantages and disadvantages of such standardized procedures are presented here in a preamble. Both users and manufacturers are encouraged to give their opinions with a view to achieving consensus on these procedures and on how further work on these lines may proceed.

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules.

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.

Thermal balloon endometrial ablation: safety aspects evaluated by serosal temperature, light microscopy and electron microscopy.

OBJECTIVES: Thermal balloon endometrial ablation is a new method for treating menorrhagia. The technique appears to be less difficult compared to standard hysteroscopic ablation techniques and to be significantly safer. The influence into the uterine wall of the thermal balloon ablation procedure was investigated with special reference to the ability of total destruction of the endometrium and the thermal action on the myometrium and the serosa. STUDY DESIGN: Temperatures were measured at the uterine serosal surface during thermal balloon endometrial ablation for 8-16 min in eight patients. After subsequent hysterectomy the extent of thermal damage into the myometrium was assessed by light and electron microscopy. RESULTS: The highest temperature measured on the uterine serosa was 39.1 degrees C. Coagulation of the myometrium adjacent to the endometrium could be demonstrated by light microscopy in all patients, with a maximum depth of 11.5 mm. By electron microscopy no influence of heat could be demonstrated beyond 15 mm from the endometrial surface. CONCLUSION: Up to 16 min of thermal balloon endometrial ablation therapy can destroy the endometrium and the submucosal layers. The myometrium is only coagulated to a depth where full thickness necrosis or injury is unlikely.

Commercial formalin substitutes for histopathology.

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200x, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.

Alveolar damage in AIDS-related Pneumocystis carinii pneumonia.

OBJECTIVE: Pneumocystis carinii pneumonia is the most common and serious of the pulmonary complications of AIDS. Despite this, many basic aspects in the pathogenesis of HIV-associated P carinii pneumonia are unknown. We therefore undertook a light and electron microscopic study of transbronchial biopsy specimens to compare pathologic features of P carinii pneumonia and other HIV-related lung diseases. DESIGN AND PATIENTS: Thirty-seven consecutive HIV-infected patients undergoing a diagnostic bronchoscopy. RESULTS: P carinii pneumonia was characterized by an increase in inflammation, edema, exudate, fibrosts, type II pneumocyte proliferation, and cellular infiltration of the alveolar wall when compared with other lung diseases (all p < 0.05). Electron microscopy showed apposition of the trophozoite to the type I pneumocyte. Erosion of type I pneumocytes was observed in 13 of 15 patients with P carinii pneumonia, whereas none without P carinii pneumonia had this finding (p < 0.05). Erosion of the type II pneumocyte was not observed. CONCLUSION: Inflammation, interstitial fibrosis, and alveolar epithelial erosion are characteristic features of P carinii pneumonia. The changes may form the pathologic basis for the respiratory failure seen in patients with P carinii pneumonia. Electron microscopy did not show any diagnostie advantage over conventional light microscopy using routine stains.

The effects of freezing, storage, and thawing on cell compartment integrity and ultrastructure.

The effects of slow freezing and thawing on enzyme compartmentalization and ultrastructure were studied in rat liver slices frozen in dry ice, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at -80 degrees C for 1-14 days. Non-frozen slices served as controls. Frozen liver slices were thawed in a Karnovsky fixative and processed for transmission electron microscopy (TEM). After all freezing protocols, the outer zone of frozen-thawed tissue was ultrastructurally very similar to that of non-frozen liver. Towards the center of the tissue, the ultrastructure progressively deteriorated. Comparison with 50-microm cryostat sections prepared for TEM showed that thawing and not freezing is the detrimental step for fair preservation of ultrastructure. After thawing, homogenization, and differential centrifugation, distribution patterns of soluble marker enzymes were analyzed (cytosol, lactate dehydrogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, acid phosphatase). The enzyme activities were not affected by storage for 2 weeks and the activity distributions showed that protein leakage from compartments was only minimally increased in frozen-thawed tissue compared with that from non-frozen tissue, irrespective of the method of freezing. In conclusion, fairly large tissue slices (20x5x3 mm) may be frozen and stored at -80 degrees C for biochemical, ultrahistochemical or ultrastructural studies. For ultrastructural analysis, only the periphery of the tissue slice should be used.

Glutaraldehyde for electron microscopy: a practical investigation of commercial glutaraldehydes and glutaraldehyde-storage conditions.

The paper takes issue with the use by glutaraldehyde suppliers of the term 'for electron microscopy', and the common practice of researchers giving insufficient or no data about the glutaraldehyde they use. Investigation of 11 commercial glutaraldehydes recommended for electron microscopy shows that only three or four of them are adequate for this purpose, using criteria set forth in papers dated between 1965 and 1989. The present paper reports that a check of purity can best be done by spectrophotometry. The 234/280 or 235/280 nm absorbance ratio is a precise indicator of the degree of polymerization, provided certain conditions stated in this paper are fulfilled. Some of the storage precautions taken by, or proposed by, suppliers are superfluous, and only mask the inadequate purification by the suppliers. A simple protocol for the storage of stock solutions is given. Alkaline glutaraldeyhyde is inherently very unstable, even in the refrigerator. Fixatives should, therefore, be stored in the freezer or should be freshly prepared.

Non-hazardous organic solvents in the paraffin-embedding technique: a rational approach. Aliphatic monoesters for clearing and dewaxing: butyldecanoate.

The aim of this study was to substitute hazardous compounds, used in tissue processing and dewaxing, with compounds having lowest possible toxicity and inflammability without impairing the morphology, staining characteristics, or diagnostic value of the tissue sections. All aromatic compounds and aliphatic hydrocarbons (e.g. alkanes, isoparaffins, petroleum distillates, etc.) were rejected, primarily due to their high vapour pressure. Based on a theoretical study of compounds used for clearing, a number of non-hazardous potential substitutes were chosen. The following experimental study narrowed the group to three unbranched, saturated, aliphatic monoesters containing 12-14 carbon atoms. On large-scale testing of these compounds, we found butyldecanoate to be the closest to an ideal substitute for aromatic and aliphatic hydrocarbons in the histology department: the section quality is at least equal to that obtained with xylene. For dewaxing, it is used at 30-35 degrees C. Butyldecanoate is not suitable as a pre-mounting agent. In practice, this is no problem as modern mounting agents permit mounting of coverslips directly from ethanol without impairing the appearance of the section in the microscope. Butyldecanoate has only a slight odour, insignificant vapour pressure (< 0.01 kPa at 20 degrees C), and does not present a fire hazard (flash point 134 degrees C). The introduction of this compound in the laboratory poses no health hazard, and the substance is biodegradable.

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.

Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure.

In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5-30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results.

Selective, quantitative detection of cadmium and/or zinc by use of BTAN (2-aminobenzothiazolylazo-beta-naphthol).

BTAN (Sumi Y et al. (1982) Histochemistry 73:481) was investigated as a histochemical Cd/Zn chelator. Cd-BTAN exhibits a main peak about 635 nm, while Zn-BTAN exhibits a main peak about 644 nm. The isobestic wavelength for Cd-BTAN and Zn-BTAN is 638 nm. The microscopical detection limit for Cd is about 25 amol/microns 2, and for Zn about 5 amol/microns 2. The relation between metal and bound chelator is fairly linear at a BTAN concentration more than 10-fold the metal concentration. Histochemical localization was fair to good, with a crystal size of up to 0.2-0.3 micron. The chelate was unaffected by hydrophilic and largely also by hydrophobic mounting media. The original staining procedure proved erratic and was modified. Posttreatment with oxine to selectively demonstrate Cd in the presence of Zn (Sumi Y et al. 1982) seriously reduced the staining intensity. Post-treatment for 8-15 min with HCl, 0.5 mol/l, in 50% ethanol removed Cd-BTAN completely with little reduction of Zn staining intensity, even from sites with 5x as much Cd as Zn. It is concluded that BTAN permits direct quantitative detection of (Zn + Cd). Provided certain precautions are taken quantitative detection of Zn and quantitation of Cd in mixed Zn/Cd sites is possible by microphotometry of the stained section before and after differentiation for 8-15 min with the HCl/50% ethanol medium.

The nutrition of the gill parasitic monogenean Pseudodactylogyrus anguillae.

A histological and histochemical study of ingested food material, energy stores and enzymes in the monogenean Pseudodactylogyrus anguillae, parasitizing the gills of the European eel (Anguilla anguilla) is presented. It was found that mucus, epithelial cells and blood from the gills were ingested. Glycogen deposits were small and primarily located in the parenchyma and to a minor extent in the vitellariae. Numerous globules of neutral lipids were found in the vitellariae. A marked esterase activity was found in the gut and a less marked activity in the vitellariae. Acid phosphatase activity was found throughout the body whereas alkaline phosphatase and leucine-amino-peptidase were not detected. Marked activity of succinate dehydrogenase and NADH-diaphorase was found in all cells, indicating a predominantly aerobic metabolism in this monogenean.

The effect of histochemical methylation on the phosphate groups of nucleic acids: interpretation of the absence of nuclear basophilia.

The effect of hot methylation (hydrochloric acid-methanol)) on nuclear stainability was investigated in order to determine whether the progressive loss of basophilia is due to methylation of the diester phosphate groups of nucleic acid. DNA spots on filter paper were unchanged in their stainability towards Toluidine Blue even after methylation for 4 days, while RNA, chondroitin sulphate and hyaluronic acid lost their affinity for this dye after 4 h methylation. In formalin-fixed sections, methylation for 4 h led to a loss of nuclear basophilia. There was no concomitant increase in nuclear relative to cytoplasmic stainability with Fast Green FCF at pH 9, as judged from the use of a comparison eyepiece, evaluation of colour transparencies or by microspectrophotometry. In contrast, extraction with trichloroacetic acid prior to or after methylation led to a much improved Fast Green staining of nuclei, comparable to the staining obtainable after treatment with trichloroacetic acid alone. In conclusion, there is no evidence that hot hydrochloric acid-methanol, as used in histochemistry, methylates the diester phosphate groups of nucleic acids. The loss of nuclear basophilia can be explained as a result of the excess positive charge on the chromatin following methylation of all the protein carboxyl groups. This effect is maximal after 3-4 h treatment with acid methanol at 60 degrees C. Further methylation leads to depolymerization and extraction of DNA. RNA is depolymerized in less than 4 h.

Aldehyde Fuchsin staining of pancreatic B cells. Reproducible high-contrast staining of formalin-fixed and paraffin-embedded material.

The use of Aldehyde Fuchsin for the demonstration of B cell granules in the pancreatic islets on material fixed in formalin and embedded in paraffin has led to variable results. Treatment of such sections for 1 h with Bouin's fluid or 5% glutaraldehyde prior to deparaffinization, however, stabilizes the secretory granules in B cells. In addition, the zymogenic granules of the acinar cells exhibit increased stainability with the permanganate Aldehyde Fuchsin procedure.

Metals and phosphate in the chloragosomes of Lumbricus terrestris and their possible physiological significance.

The chloragog cells of the earthworm Lumbricus terrestris contain numerous granules, chloragogomes, which were analyzed for metals and phosphate by histochemistry, by use of an electron microscope X-ray microprobe (EMMA) and by chemical analysis of chloragosome preparations. Inorganic and organic phosphate each accounts for about 3% of the chloragosome dry mass, Ca for 2--3%, Zn for 1--3% and Mg for 0.2--0.4%. Carbonate is not present in chloragosomes. The average molar ratios Ca:Mg:Zn:total PO4 are 1:0.1:0.3:1. The Ca:PO4 ratio is fairly constant (correlation coefficient 0.99), while the Zn:PO4 ratio varies considerably. It is concluded that Ca is bound in the form of inorganic CaHPO4 and organic R-OPO3Ca (or possibly Ca-polyphosphate complexes). Mg may also be phosphate-bound, while Zn probably is not. Chemical analysis of the calciferous glands revealed a high concentration of Ca, small amounts of Mg and phosphate, but no Zn. It is concluded that Zn is not excreted through the calciferous gland. Storage of Ca in the chloragocytes and excretion of CaCO3 by the calciferous gland may be physiologically linked. Regulation of the concentrations of Ca and HCO3- ions in the blood and coelomic fluid may assist in equalization of osmotic pressures during dehydration and rehydration. This regulation may be a major function of the chloragosomes. The chloragosomes were discussed in relation to the "spherites" of various arthropods and molluscs and to the "cytosomes" of anoxia-tolerating molluscs.