Eosin interaction of alpha-synuclein leading to protein self-oligomerization

Among various dyes including congo red, thioflavin S, thioflavin T, eosin, rhodamine 6G, and phenol red, the eosin was the only dye that induced self-oligomerization of alpha-synuclein in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline. To analyze chemical nature of the eosin interaction with alpha-synuclein, the phenomenon of self-oligomerization was further examined with eosin congeners such as ethyl eosin, eosin B, phloxine B, erythrosin B, and rose bengal. The followings are the conclusions we have reached. First of all, intactness of the benzoate moiety of eosin and the negative charge on the carboxylic group of the dye are important factors leading to the specific interaction with alpha-synuclein. Secondly, the localized negative charge on the xanthene moiety of eosin is another critical factor for the interaction. As far as substituting halides are concerned, bromides and iodides on the xanthene moiety of the dyes do not make any difference on the alpha-synuclein interaction because both eosin and erythrosin B have induced the common phenomenon of self-oligomerization. The binding curve between eosin and alpha-synuclein was sigmoidal as the dye concentrations were increased. A double reciprocal plot of the saturation curve showed that the maximum number of eosin binding sites on alpha-synuclein was 1.85 with a dissociation constant of 390 microM. The dye binding to the protein appeared to occur via a positive cooperativity. The eosin binding site(s) was suggested to be located predominantly on the NAC region and partly related to the acidic C-terminus of alpha-synuclein. It has been, therefore, expected that this information might be useful to develop alpha-synuclein interactive molecules, which could provide eventual preventive or possible therapeutic means against various alpha-synuclein related disorders including Parkinson's disease.     

Eosin staining of gelatine

Summary The mechanism of gelatine staining with four selected fluorone derivative dyes (eosin y, ethyl eosin, methyl eosin, uranin) was investigated. Gelatine films were stained in dye-buffer-ethanol solutions at varying pH and in the presence of NaCl and urea. Dye binding was recorded spectrophotometrically. Ionization constants of auxochromic phenolic groups were determined from pH-absorbance curves of dye-buffer-ethanol solutions. Dyebinding was greatest at pH below pK_OH and decreased with increasing pH. The addition of NaCl reduced dye binding slightly below pK_OH but markedly above pK_OH. The addition of 8 M urea decreased dyebinding regardless of pH. Comparing the pH dependence of dyebinding for eosin y and esterified eosins with ionization constants revealed that ionic bonding is unlikely to occur at the carboxyl group as well as at the phenolic group. Dye binding is intimately related to the presence of Br-groups. These results are discussed in conjunction with the functional structure of the dye ions and current concepts of dyebinding mechanisms.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

Staining Problems with Eosin Y: A Note from the Biological Stain Commission

In 1980, eosin Y was the certified dye with which technologists encountered most problems. The specific problem most frequently brought to the attention of the Biological Stain Commission was that solutions of eosin Y formed a precipitate and failed to stain cytoplasm red when used as a counterstain to hematoxylin.     

Standardized Thionin-Eosin Y: A Quick Stain for Cytology

Two stains long used in exfoliative cytology, the hematoxylin-eosin Y and Papanicolaou stains, have not been standardized even today. Some dozens of hematoxylin and eosin and Papanicolaou staining recipes have been recommended in the literature. Consequently, the staining pattern of hematoxylin and eosin, and Papanicolaou stained cytological material varies from laboratory to laboratory. To a certain degree this is due to batch-to-batch variations of commercial samples of the natural dye hematoxylin (C.I. 75290). The present paper describes a simple, standardized and reproducible procedure using thionin bromide to replace hematoxylin in the hematoxylin and eosin stain.     

Improved nuclear staining with mordant blue 3 as a hematoxylin substitute.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely results very resemble a good hematoxylin and eosin.     

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

Improved Nuclear Staining with Mordant Blue 3 as A Hematoxylin Substitute

For progressive staining 1 g mordant blue 3, 0.5 g iron alum and 10 ml hydrochloric acid are combined to make 1 liter with distilled water. Paraffin sections are stained 5 minutes, blued in 03% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely resemble a good hematoxylin and eosin.     

Quirks of dye nomenclature. 10. Eosin Y and its close relatives

The long history of eosin Y, eosin B and the methyl and ethyl eosins is recounted as well as their synthesis, the variety of their molecular species and some of the myriad applications of these dyes. Chromatographic techniques are described that reveal the purity or lack of it in commercial samples. Toxicological studies are discussed that suggest that the eosins are virtually non toxic, but efforts to remove them from the environment imply that there may be some risk.     

Mordant Blue 3: a Readily Availablx Substitute for Hematoxylin in the Routine Hematoxylin and Eosin Stain

Mordant blue 3 may be used as a suhstitute for hematoxylin in hematoxylin and eosin stains. The staining solution consists of 0.25 g dye, 40 ml of 10% iron dam, 5 ml of cone H₂SO₄, and 955 ml of dirtilled H₂O. Staining the is 5 minutes, followed by differentiation in acid water or acid alcohol. After blueing, the seaions are counterstained with emin. Results closely resemble the hematoxylin and eosin stain.     

History of Staining the Staining of Blood and Parasitic Protozoa

By the term “blood stain” one ordinarily means a compound dye formed from the chemical union of an acid and a basic dye, and usually a compound of the eosin-methylene-blue group. It is well known today that the sodium salt of a color acid (e. g. eosin) and the chloride of a dye base (e. g. methylene blue) may be converted by simple metathesis into sodium chloride plus the compound dye (e. g. methylene blue eosinate), the latter being insoluble in water unless an excess is present of either the acid or the basic dye. In modern blood stains a compound dye of this type is dissolved in methyl alcohol and mixed with water on the slide at the moment of staining.     

Mechanisms involved in the chemical inhibition of the eosin-sensitized photooxidation of trypsin

A large series of compounds was screened for ability to protect trypsin from eosin-sensitized photodynamic inactivation. Eosin-sensitized photooxidation reactions of this type typically proceed via the triplet state of the dye and often involve singlet state oxygen as the oxidizing entity. In order to determine the mechanisms by which trypsin is protected from photoinactivation, a number of good protective agents (inhibitors) and some non-protective agents were selected for more detailed flash photolysis studies. Good inhibitors such as p-phenylenediamine, n-propyl gallate, serotonin creatinine sulfate and p-toluenediamine competed efficiently with oxygen and with trypsin for reaction with the triplet state of eosin. The inhibitors were shown to quench triplet eosin to the ground state and/or reduce triplet eosin to form the semireduced eosin radical and an oxidized form of the inhibitor. In the latter case, oxidized inhibitor could react by a reverse electron transfer reaction with the semi-reduced eosin radical to regenerate ground state eosin and the inhibitor. The good inhibitors also competed effectively with trypsin for oxidation by semioxidized eosin, thus giving another possible protective mechanism. Non-inhibitors such as halogen ions and the paramagnetic ions Co++, Cu++ and Mn++ reacted only slowly with triplet and with seimioxidized eosin. The primary pathway for the eosin-sensitized photooxidation of trypsin at pH 8.0 involved singlet oxygen, although semioxidized eosin may also participate.     

Staining Etched Epoxy Resin Sections for Light Microscopy

Staining of etched sections for light microscopy is described. Azan staining was successful after treatment with potassium dichromate and the use of concentrated dye solutions. To remove osmium for hematoxylin-eosin staining, removal by reduction with ferrocene was used instead of oxidation. Highly selective differentiation after hematoxylin staining was achieved using p-toluenesulfonic acid-DMSO. To enhance eosin staining, a 2-bromoethylamine link between eosin and the tissue was used. Ferrocene also facilitated counterstaining of nuclei with hematoxylin after the PAS reaction. Periodic acid-methenamine silver staining was carried out without modification.     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

Eosin B

The two known isomeric dibromodinitrofiuoresceins have been prepared in a fairly pure state, and their absorption spectra determined.

Commercial samples of eosin B are not 4, 5-dibromo—2, 7-dinitrofluorescein, as stated in dye indices. They are mixtures which contain other bromonitro derivatives of fluorescein as well as di-bromodinitro derivatives.

The color acid method provides a substantially reliable means of determining actual dye content with commercial samples of the dye, but the reduction method may prove decidedly misleading.     

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.