The composition of stains produced by the oxidation of Methylene Blue

Synopsis The composition of some stains produced by the oxidation of Methylene Blue has been studied by thin-layer chromatography. Various named methods for the production of Polychrome Methylene Blue, Azure A, Azure B, Azure C and Methylene Violet Bernthsen have been found to give complex mixtures of varying proporitions of up to eleven dyes. Ten of these, namely Methylene Blue, Azure B, Azure A,sym-Dimethylthionine, Azure C, Thionine, Methylene Violet Bernthsen, Methyl Thionoline, Thionoline and Thionol, have been identified by their visible absorption spectra. The remaining dye could not be identified. When used on a laboratory scale, these methods give stains of constant composition independent of the batch of Methylene Blue. Stain composition as revealed in the present study has been compared with that previously indicated by other, less effective, analytical techniques. Reasons are presented why the latter give equivocal results.     

Acid mucopolysaccharides in hair papillae of the stump-tailed macaque (Macaca speciosa).

Synopsis Acid mucopolysaccharides in dermal papillae of hair follicles from both bald and non-bald regions of the scalp of stump-tailed macaques were studied histochemically. Alcian Blue, Azure A and Periodic acid Schiff methods were used for staining mucopolysaccharides, and Bromphenol Blue for staining basic proteins. In an attempt to identify various polyanions, staining was carried out with Alcian Blue containing different concentrations of electrolytes. Methylation, saponification, mild acid hydrolysis and digestion with streptomyces or testicular hyaluronidase, chondroitinase ABC, or sialidase, were also used. The results indicate that chondroitin sulphate B is present in the papillae of terminal hair follicles in early and intermediate anagen, and degraded chondroitin sulphates are present in the papillae of vellus and terminal hair follicles in late anagen.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Summary Four methods based upon the periodic acid—phenylhydrazine—Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizablevicinal diols (hexose, 6-deoxyhexose,N-acetylhexosamine) and either sialic acids or side chainO-acyl sialic acids. In the first of these procedures, the saponification—periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification (KOH—PA—DNPH—Az—KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizablevicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A Schiff—saponification (PA—DNPH—Az—KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have anO-acyl substituent at position C7, while in the third method, the selective periodate oxidation—borohydride reduction—saponification—periodic acid oxidation—2,4-dinitrophenyl hydrazine—Azure A—Schiff—saponification (PA^*—Bh—KOH—PA—DNPH—Az—KOH) technique, only sialic acids withO-acyl substituents at positions C7, C8 or C9 (or which have two or threeO-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification—borohydride reduction—periodic acid oxidation—Schiff (PA—DNPH—Az—KOH—Bh—PAS), sialic acids without side chain substituents or which haveO-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.     

Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells

Synopsis Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340/394 nm) and emission (443/700 nm) curves.     

Exploration of dye chemistry in Taenzer Unna Orcein type elastin staining

Summary Musso's demonstration of the amino- and hydroxyphenoxazone structure of synthetic orcein suggested trial of simpler mixtures and isolated pure dyes of the phenoxazine phenoxazone series. It was further thought that explorations of this sort might reveal which of the hitherto demonstrated 14 chromatographic components of orcein might take part in or even be chiefly responsible for the elective staining of elastin.Accordingly trials were made in a hydrochloric acid (0.12 N) 70% alcohol technic based on Taenzer's original method of a number of commercially available dyestuffs and indicators and a number of products were synthesized by variants of Musso's technic for resorcin blue: air oxidation of resorcinol in the presence of ammonia. Those tested are the following: Azolitmin, Lacmoid, Resorufin, Resorcin Blue (MLB), C.I. No. 51020, Gallocyanin C.I. 51030, Brilliant Cresyl Blue C.I. 51010, Nile Blue C.I. 51180, a Nile red preparation, Bernthsen's Methylene Violet; Azure A, Toluidine Blue C.I. 52040, several laboratory synthetic lots of Musso's Resorcin Blue in which oxidation was done with H₂O₂ and varying amounts of ammoni were used, and 2 batches in which resorcinol was partly or completely replaced by m-aminophenol.Successful to excellent elastin stains were achieved with Lacmoid, Resorcin Blue MLB, part of the Musso Resorcin Blue products and the two m-aminophenol oxilation products Elastin purple FP and Elastin Videt PR.From the failure of azolitmin and resorufin, both 7-hydroxy-2-phenoxazones and the success of resorcin blue (MLB) 7-N,N-dimethylamino-2-phenoxazone, it appears suggested that the aminophenoxazone structure may be a determining characteristic. The success with m-aminophenol substitution in the Musso air oxidation NH₃ synthesis tends to support this view.While I have had some success with the Victoria blues first used by Lustgarten, using other technics, these dyes and some other triphenylmethanes do not successfully take the place of orcein in acid alcohol staining methods. Rosanilin and pararosanilin do stain rodent elastica from acid aqueous and alcoholic solutions but adult human elastica does not so stain. We suspect a diphenamine Schiff base condensation with the known free aldehyde of rodent elastica. This is confirmed by more or less complete blockade of pararosanilin acid alcohol staining with p-toluidine in glacial acetic acid, 1 hr, hydroxylamine: sodium acetate: H₂O 102040 3 hr and 5% phenylhydrazine HCl 3 hr on dog, rat and guinea pig arteries. The hydroxylamine gave complete blocking, the other two reagents partial. Altogether about 50 dye samples were tested.Presented before the Histochemische Gesellschaft September 28, 1968.Supported by National Cancer Institute Research Grant C-4816, National Institutes of Health.     

On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azures B-Eosin Y stain as compared with other thiazine dye-Eosin Y combinations

Summary After incorporation into a polyacrylamide matrix, the biopolymers DNA, RNA, heparin, hyaluronic acid, collagen and the synthetic polymers poly(U) and poly(A, U) were stained with the pure thiazine dyes, Methylene Blue, the Azures and Thionin alone and combined with Eosin Y. Satisfactory spectrophotometric agreement was obtained between the staining reactions of the biopolymers in the artificial matrix and those in their natural surroundings. This was especially true with respect to the specificity of the Azure B-Eosin Y dye-pair, which is based on the generation, on suitable substrates, of a purple colour, the Romanowsky-Giemsa effect (RGE), with an absorbance maximum near 550 nm. In the model experiments, DNA, heparin, hyaluronic acid and collagen were found to be RGE-positive and poly(U), poly(A, U) and RNA RGE-negative.A theory of RGE is proposed which complies with the new and earlier observations: after saturation of available anionic binding sites and aggregate formation by Azure B, electron donor acceptor complexes are formed between Eosin Y and Azure B via hydrogen-bridge formation of the aminosubstituent proton of Azure B and between Eosin Y and the biopolymer surface. Charge-transfer complex formation may also account for the qualitative identity of Azure B-Eosin Y and Azure A-Eosin Y spectra of substrates, which are coloured purple. Quantitatively, Azure A-Eosin Y is less efficient in giving RGE. The generation of RGE is time-dependent. Equilibrium staining is attained after about 120 h. The implications of the results for the biological application of Romanowsky-Giemsa staining are discussed briefly.     

Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

A new method for myofibrillar Ca++-ATPase reaction based on the use of metachromatic dyes: its advantages in muscle fibre typing

A new method for Ca++-ATPase reaction in human muscle fibres is presented as an alternative to previous ATPase stains. The method is based on the use of metachromatic dyes, namely Azure A and Toluidine Blue, and has the advantages of speed, ease of performance and production of an elegant and clearcut fibre typing. The method distinguishes fibre types because of their metachromatic or orthochromatic staining, due to their different content of phosphate after incubation in the reaction medium. The comparison of serial sections stained by cationic dyes and by ammonium sulphide revealed close correspondence of fibre typing. Fibre type differentiation was also obtained with Acridine Orange; however this method was less reproducible.     

The Giemsa-11 technique for species-specific chromosome differentiation

Summary Oxidizing Methylene Blue and adding the reaction products to Eosin Y and Azure B makes possible a highly reliable Giemsa-11 technique for discrimination of chromosomes in hybrid cells according to their parental origin. This staining can be combined in a sequential procedure with a fluorescent banding technique allowing the exact identification of the chromosomes.     

A puzzling feature of Colloidal Iron positive and Alcian Blue negative material

Summary A puzzling feature of Colloidal Iron positive and Alcian Blue negative substance is encountered in yolk sac of young larvae of a fish —Tilapia mossambica. This yolk material is PAS positive (proved to be due to neutral mucopolysaccharide) and negative to Toluidine Blue, Azure A (pH 2 to 4.5), Aldehyde Fuchsin and AB pH 1. More work is necessary to establish the exact chemical nature of the CI positive material.     

Paraphyly of the Blue Tit (Parus caeruleus) suggested from cytochrome b sequences

The phylogenetic relationships of the Blue Tit-Azure Tit assemblage (genus Parus; Aves: Passeriformes) were studied using mitochondrial DNA sequences of 24 specimens representing seven subspecies from Eurasia and North Africa. Previous work based on comparative morphological and acoustic data suggested a division of the Blue Tit (Parus caeruleus) into two species. Our analyses clearly indicate that the Blue Tit represents a paraphyletic assemblage, including a European/Middle Asian clade that is the sister group to the Azure Tit (Parus cyanus) and a North African clade. The North African clade (teneriffae subspecies group) is a sister group to the European Blue Tit/Azure Tit clade. We suggest a division of the Blue Tit into two separate species, Eurasian Blue Tit (Parus caeruleus s. str.) and African Blue Tit (Parus teneriffae). However, our data give no support for assigning species rank to Parus cyanus flavipectus, a subspecies of the Azure Tit, as suggested by several authors on morphological grounds.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Effects of anhydrous benzoylation on staining with a variety of protein end-group methods

Summary The effects of anhydrous benzoylation were examined in connection with the following protein end-group methods: diazosulfanilic acid — Azure A, the Sakaguchi reaction and a fluorescent variant, the phenanthrequinone fluorescent method for arginine, the Millon reaction, the Morel-Sisley reaction, the mercury orange method, the alloxan and ninhydrinShiff methods, the Dansyl-chloride fluorescent method for lysine, the dimethylaminobenz-aldehydenitrite (DMAB) method for tryptophan, and the acid fluorochrome brilliant sulfoflavin used at pH 2.8 as a stain for basic groups. With all of these methods except the DMAB method for tryptophan, selective blockage of cytoplasmic proteins and staining of nuclei was obtained. With the DMAB reaction, the results were reversed: cytoplasmic staining exceeded nuclear staining after benzoylation.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Cytotoxicity, radiosensitization, and DNA interaction of platinum complexes of thiazin and xanthene dyes

Complexes of the platinum(II) tetrachlorodianion with positively charged nuclear dyes have been prepared in an effort to produce neutral molecules which could gain ready access to the nuclear DNA where the platinum(II) tetrachlorodianion could function as a radiosensitizing and a bifunctional alkylating agent. The thiazin dyes Thionin, Azure B, and Methylene Blue, the aminoxanthene dye Pyronin Y, and the thiazole dye Thioflavin have each been complexed to the platinum(II) tetrachlorodianion(PtCl4) in a ratio of 2:1(dye:PtCl4). Studies of the interaction of these complexes and of the dyes with the pBR322 plasmid superhelical DNA demonstrated that while each complex and dye readily associated with the DNA in a dose-dependent manner, only Pt(Thioflavin)2 and Thioflavin produced irreversible DNA changes (single-strand breaks). In exponentially growing EMT6 cells the cytotoxicity of these drugs was assessed in normally oxygenated and hypoxic cells at both pH 7.4 and 6.45. At concentrations ranging from 1 to 500 microM, Pt(Methylene Blue)2 was significantly more cytotoxic than the other thiazin dye complexes Pt(Thionin)2 and Pt(Azure B)2. The cytotoxicity of Pt(Thionin)2 and Pt(Methylene Blue)2 was increased in normally oxygenated and hypoxic cells at low pH. Both Pt(Pyronin Y)2 and Pt(Thioflavin)2 were more toxic than the thiazin complexes. Pt(Pyronin Y)2 was most cytotoxic to normally oxygenated cells at normal pH and hypoxic cells at low pH, while Pt(Thioflavin)2 was most cytotoxic to cells at low pH under both oxygenation conditions. In vitro studies of the radiosensitizing properties of these agents in EMT6 cells demonstrated that exposure to 100 microM for 1 h before and during irradiation (except for Pt[Thioflavin]2, which was assayed at 25 microM) resulted in enhancement rations of 2.5, 1.9, 1.5, and 1.5 for Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y)2, and Pt(Thioflavin)2, respectively, in hypoxic cells. In contrast, Pt(Methylene Blue)2 (and Methylene Blue) proved to be a radioprotector of normally oxygenated cells and did not sensitize hypoxic cells to the cytotoxic effects of radiation. In the FSaIIC fibrosarcoma in vivo administration of each drug at 100 mg/kg intraperitoneally (ip) 15 min prior to irradiation (except for Pt[Thioflavin]2, which was given at 1 mg/kg ip) showed that, with single radiation fractions of 10 and 20 Gy, dose-modifying factors of 2.1, 1.8, 1.5, and 1.2 were produced by Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y), and Pt(Methylene Blue)2, respectively, after correcting for growth delays induced by the drug alone. In comparison, misonidazole at 1 g/kg ip produced a dose-modifying factor of 1.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.     

Quantitative microspectrophotometry of RNA in plant tissue

Synopsis Chemical estimation of nucleic acid, essentially RNA, in fixed tissue from Jerusalem artichoke tubers, coupled with an examination of the types of RNA in the fixed tissue by gel electrophoresis, demonstrates that ribosomal and soluble RNA are preserved in this tissue after various fixation procedures including methanol, ethanol-acetic acid and aqueous formaldehyde. Tissue fixed in ethanol-acetic acid or formaldehyde is resistant to loss of nucleic acid by aqueous extraction but tissue fixed in all three standard fixatives loses nucleic acid in citrate buffer under conditions used for Azure B staining. The presence of Azure B in the buffer does not wholly prevent this loss. Tissue fixed in formaldehyde or mixed fixatives containing formaldehyde is resistant to loss of nucleic acid during treatment with EDTA to obtain cell suspensions.Preliminary experiments with Azure B, Gallocyanin chrome-alum and Methylene Blue showed that the Gallocyanin technique is the most practicable for demonstrating RNA cytochemically. Its specificity was confirmed by ribonuclease extraction of the tissue. Optimum staining conditions, requiring treatment of the tissue in Gallocyanin chrome-alum solution overnight at 40°C, are established for the artichoke tissue and for paraffin sections of pea shoot apices.     

Mucopolysaccharide histochemistry of Musca domestica

Summary While studying the distribution of acid mucopolysaccharides (AMPS) in the prepupal larva of the housefly, Musca domestica nebulo (Fahr.) using routine histochemical methods, the authors found that except for the midgut cells, dorsal pharyngeal epithelium and the thick connective tissue of the brain, the ganglia and the imaginal discs, in all other cells studied, an AMP is present which is neither a sulfomucin, a sialomucin nor a hyaluronic acid. Its alcian blue (AB) staining is abolished by active methylation and is not restored by saponification; it is not abolished by mild acid hydrolysis; it stains -metachromatically with Azure A at pH 4.5 and lightly with Azure A at pH 1.5, with weak toluidine blue and with aluminium sulfate-methylene blue; it is unstained by aldehyde fuchsin (AF) and safranin O (Saf. O) in AB-AF, AF-AB and AB-Saf.O procedures; it is positive to colloidal iron but is negative to periodic acid Schiff reaction in all probability; and its AB staining is abolished by saponification (KOH treatment) without prior methylation. Pending further work to exactly characterize this AMP, it is tentatively referred to as KOH-labile AMP. The significance of its distribution is discussed.