The interference of L-ascorbic acid with a standard histochemical azo dye coupling procedure

Summary The reducing agent, L-ascorhic acid, has been shown to interfere with the chromogenesis of free Napthol AS D, and the diazonium salt, fast red violet.     

A simultaneous-coupling azo dye method for the quantitative assay of esterase using alpha-naphthyl acetate as substrate

Summary This paper describes a simultaneous-couplng azo dye method for the measurement of estarase activity using the histochemical substrate, -naphthyl acetate. By the choice of two diazonism salts with optimal coupling characteristics, the reaction be carried out at any pH between 3.0 and 9.5. The azo dye is maintained in solution for spectrophotometric measurements with bovine serum albumin. The simultaneous-coupling method is compared with an assay based on the direct measurement of released -naphthol by its ultra-violet bsorbance in a pH study of hog liver esterase. There is good agreement between the data obrained by both methods.     

An X-ray microanalytical azo dye technique for the localization of acid phosphatase activity

Summary A new method is described for the histochemical localization of acid phosphatase. Naphthol AS BI, enzymatically released from naphthyl AS BI phosphoric acid, is coupled with diazotized 2,5-dibromoaniline to produce a fine insoluble red azo dye. The histochemical and cytochemical localization of this final reaction product in rat liver is described. In the electron microscope, sites of the azo dye can be detected by X-ray microanalysis of ultrathin cryosections of reactive tissue.This research was supported by Scientific Research Council Grant No. B/RG/67527     

A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.     

A technique for quantitative estimation of small mammals traversing water obstacles

A new method for quantitative study of small mammals swimming across water obstacles was developed. A line of 25 traps was installed on either poles or boards (“rafts”) with anchors at a distance of 20–25 m from the bank and with 10-m distances between the items. The study was performed upstream along the Ilych River in August 2013. A total of 300 trap/day were accumulated. Twenty-four small mammal individuals of 8 species were captured. Their relative abundance was estimated as the number of individuals per 100 trap/day. It was found experimentally that floating poles neither repel nor attract animals. When an individual accidentally finds a floating pole, it climbs up and explores it for some time. The number of animals per total length of rafts per time unit can be suggested as an index of intensiveness of migration across a water obstacle. In the area studied, the number of small mammals of various species crossing the river was estimated at 26.7 individuals per 1 km/day. A length of 5 m for floating poles/boards and installation of two traps at the ends of an item is suggested to be used.     

Are picro-dye reactions for collagens quantitative? Chemical and histochemical considerations

Previous studies of picro-dye reactions demonstrated wide variations in the binding of different dyes. Picro-Sirius Red F3BA was recommended because it colors all collagens intensely and is suitable for polarization microscopy. Recent publications on quantitative uses of this stain were surprising. To obtain further information on the chemical mechanisms of dye binding by proteins, 94 sulfonated azo dyes were tested under the conditions of the picro-Sirius Red F3BA reaction. Reaction patterns varied widely, from failure to compete successfully with picrate ions for binding sites to strong coloration of all tissue structures. Only a few dyes stained collagen, reticulum fibers and basement membranes intensely and selectively. The reactivity of dyes was determined by their molecular configuration and the nature and position of substituents. Correlation with physico-chemical data showed that dye binding is due to non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding. Coulomb forces do not impart affinity - increasing sulfonation actually decreases dye uptake - but draw dyes within reach of non-ionic sites. Bound dyes form aggregates with additional dye ions; the aggregation number can range from 2 to many powers of 10. Clearly, dye binding by proteins is not stoichiometric.     

Reduction and azo coupling of quinones. A histochemical study of human cutaneous melanin and adrenochrome.

Cutaneous melanin in formol fixed skin and adrenochrome in dichromate fixed monkey adrenal after adequate bisulfite or dithonite reduction were found to give definite azo coupling reactions. Weaker reactions were obtained on unreduced material, and these disappeared on ferric chloride oxidation. Both cutaneous melanin and adrenochrome appear to exist in a quinhydrone status. Prolongation of dichromate treatment weakens or abolishes azo coupling capacity of adrenochrome. The findings support the concept of quinonization and reduction to prevent and restore azo coupling of enterochromaffin cells and noradrenaline islets of the adrenal. The most effective diazos for melanin were p-nitrodiazobenzene, fast black K and the diazosulfanilic acid, pH 1 pyronin B procedure, for adrenochrome. Diazosafranin and 2-chloro-4-nitrodiazobenzene were also useful. Blue and violet coupling products from toluidine blue and methylene violet RR fail to yield sufficient contrast to be convincing.     

Tartrazine: solid-phase radioimmunoassay studies of an azo dye implicated in allergic reactions (azo dyes and allergy).

A solid-phase radioimmunoassay procedure was adapted for the haptenic study of tartrazine, an azo dye implicated in various forms of allergy. Further, the haptenic relationship of tartrazine and aspirin was investigated, since sensitivity of individuals to the two substances is often clinically associated. The specificity of antibody to tartrazine was directed strongly toward a pyrazolone intermediate of the molecule, 1-(4-sulfophenyl)-3-carboxy-5-hydroxy-pyrazole. Aspirin did not cross-react with anti-tartrazine, suggesting that the clinical association of aspirin and tartrazine sensitivity in patients is a nonimmunological phenomenon.     

The histochemical demonstration of gamma-glutamyl transpeptidase activity in different populations of rat liver during azo dye carcinogenesis

To better assess the reliability of gamma-glutamyl transpeptidase (gamma-GTase) as a marker of preneoplastic liver lesions and hepatomas, the gamma-GTase activity of different cell populations was examined in liver sections from rats fed 4-dimethylaminoazobenzene. The results indicated that the biliary ductular cells in trabeculae of cirrhotic livers may exhibit appreciable gamma-GTase activity in addition to that shown by islands of regenerating parenchyma. At later stages of azo dye carcinogenesis, the epithelial cells of bile duct cysts and cholangiomas, as well as those of hepatomas, gave positive reactions for gamma-GTase. Thus biochemical data on liver gamma-GTase in different models of hepatocarcinogenesis cannot be translated directly in terms of alterations in a particular cell type unless such interpretation is justified by parallel histochemical investigations.     

A quantitative electron histochemical estimation of ruthenium red binding to heparin in mast cell granules

Summary Mast cell granules were shown to contain an apparently branched meshwork of fibers, which had a diameter of about 240 Å and a denser core of about 20–30 Å. Mast cell granules from rats were found to have a median weight of 39×10^–14 g after critical-point drying. Their dry mass increased 23% when stained with ruthenium red at pH 5.0. The staining reaction was found to be stoichiometric, and the bulk of the stain appeared to be taken up by heparin in a molar relationship of one ruthenium red cationic complex per sulfate group of heparin. The uptake of the stain was largely localized to the cores of the granule fibers, which after staining appeared double contoured. These findings suggest that mast cell heparin is integrated into the fibrous structure of the mast cell granules.This project was supported in part by Grant No. P-259-H from the American Cancer Society.The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.