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R W Horobin

Publications and source records attributed to R W Horobin.

At least 55 records · Page 3Linked to original sources
PubMed1986

Prediction of in situ fluorescence of histochemical reagents using a structure-staining correlation procedure.

A total of ninety acid, basic, and non-ionic dyes were screened for fluorescent staining of various Carnoy fixed rat tissues. It was found that the fluorescence/non-fluorescence of a dye could be predicted using a conjugated bond number (CBN) cut-off value. Thus 90% of dyes with CBNs of 29 or less were fluorescent; whilst 70% of dyes whose CBNs exceeded 30 were nonfluorescent. The cut-off value was not significantly influenced by the charge, or the hydrophobic-hydrophilic character of the dye; though fluorescence was greatly influenced by the mode of fixation. The CBN cut-off value proved surprisingly robust. Thus most fluorochromes found in the histochemical literature have small conjugated systems, with CBNs less than the cut-off value. This includes labels of immunoglobulins, vital stains of neurones, and fluorescent Schiff reagents. Conversely several dyes used to quench background autofluorescence have large conjugated systems, with CBNs substantially above the cut-off value.

Animals↗
PubMed1985

A widely applicable analytical system for biological stains: reverse-phase thin layer chromatography.

The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. Rf values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.

Chemical Phenomena↗
PubMed1984

Novel application of aniline blue. Fluorescent staining of glycogen and some protein structures.

The present study shows that aniline blue can be used as a fluorescent stain for glycogen. The dye is also helpful in tracing pathological and autolytic changes in lysosomes, mitochondria, erythrocytes and nuclei, and it can also be used for demonstrating bacteria in tissue sections and smears. The techniques used are simple, rapid and inexpensive. Spectrophotometric studies on aniline blue solutions have shown that aniline blue fluorescence was enhanced by the addition of certain proteins, or of glycogen to the dye solution. In case of albumen which has the maximum effect, enhancement is dependent upon the albumen-dye ratio. The mechanism of staining is mainly due to self quenching, but there is also an evidence of the presence of hydrophobic reaction.

Aniline Compounds↗
PubMed1983

Variation between different samples of SITS with respect to axonal transport and toxicity.

Retrograde axonal transport of the fluorescent compound SITS has been described as occurring only from axon terminals and not from axons of passage. Injection of 4 different commercially available samples of SITS into terminations of cerebellar pathways in the rat revealed that only one sample produced retrogradely labelled neurones. Chemical analysis suggested that this was due to a unique fluorescent component (not SITS). This sample also contained other fluorescent components one of which, present in two other samples, produced paroxysmal motor disturbances. All of the samples examined contained several other fluorescent compounds.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1983

The aging of Gomori's aldehyde-fuchsin: the nature of the chemical changes and the chemical structures of the coloured components.

The chemical nature of Gomori's aldehyde-fuchsin (GAF) prepared by the method of Mowry et al. (1980) was studied by monitoring the staining characteristics and various chemical properties of ripening GAF solutions. Hydrophilic-hydrophobic characteristics were assessed by reverse-phase thin-layer chromatography; overall molecular sizes, and also the sizes of the conjugated systems, were investigated using hydrophobic gel-filtration. Other properties monitored included the nuclear magnetic resonance and visible spectra, the apparent pH, and the amount of precipitation. It was concluded that for GAF prepared by the Mowry et al. procedure: i. The purple, strongly staining components arising in the first few days of aging are Schiff bases derived from Pararosanilin. ii. These Schiff bases are slowly transformed into a variety of N-ethylated derivatives, which are weakly or non-staining. In addition to these chemical transformations, the quality of staining was markedly influenced by the tendency of GAF to precipitate; as much as three quarters of the dye was sometimes lost in this way during the first few days or weeks.

Animals↗
PubMed1983

Staining plastic sections: a review of problems, explanations and possible solutions.

Many of the difficulties of staining plastic embedded tissues for light and electron microscopy derive from physical exclusion of hydrophilic staining reagents by hydrophobic embedding media. Structures which stain most intensely with hydrophilic reagents usually contain less hydrophobic plastic than do non-staining structures. Such incomplete infiltration is apparently caused by exclusion of viscous, hydrophobic monomers by physically dense and/or well hydrated tissue elements. In keeping with this, generalized staining of tissues embedded in hydrophobic media does occur when hydrophobic reagents are used. Staining of plastic-free structures with single hydrophilic reagents or with sequences of such reagents, is, however, largely rate-controlled. The surprising similarity of hydrophilic and hydrophobic plastic embedding media is discussed. Limits of this simple model are explored, with a consideration of the roles of fixative and of monomer-tissue reactions.

Histological Techniques↗
PubMed1982

Chromosome banding: specification of structural features of dyes giving rise to G-banding.

Metaphase chromosomes were stained in a routine G-banding procedure with 39 basic dyes of varied structures substituted for the Giemsa stain. Staining outcomes were categorized as: overstained, differentially stained, trivially or unstained. Certain structural features of the dyes were described numerically, namely, largest conjugated fragment (LCF), conjugated bond number (CBN) and cationic weight. The staining outcomes were compared to these numerical structural parameters, and structure--staining correlations sought. Dyes with large conjugated systems (and high LCF values) were seen to be overstained; dyes with low LCF values were often non-staining. At intermediate LCF values, the more hydrophobic dyes (with high Hansch pi values) stained differentially; the more hydrophilic dyes failed to stain. Expressed numerically, 89% of the dyes with the following characteristics stained differentially: 30 greater than or equal to LCF greater than or equal to 10; Hansch pi greater than -5.0. It was concluded that contributions to dye-chromosome affinity included coulombic forces and van der Waals attractions and that the selectivity of G-banding was largely due to hydrophobic bonding. Induction of bands could be due to the loss of hydrophilic histones, amplifying underlying variations in the hydrophobic-hydrophilic character of the chromosome structure. Relatively hydrophobic sites include AT-rich DNA and disulphide-rich proteins. The effects on Romanowsky G-banding of chemically modifying chromosomes were in keeping with this model. Overstaining resulted from formation of either hydrophobic or conjugated derivatives or both, whereas trivial or non-staining arose from the formation of hydrophilic derivatives. Intriguingly, the efficacy of the dyes used for Q-banding also correlated positively with their hydrophobic character.

Chromosome Banding↗
PubMed1982

Selection of optimum tetrazolium salts for use in histochemistry: the value of structure-staining correlations.

A structure-staining correlation study was made of a number of tetrazolium salts and formazans used in histochemistry. Numerical parameters describing molecular structure were calculated from structural formulae. Staining data were from previously published work. It was found that several significant practical variables (namely, uptake and reducibility of tetrazolium salts; substantivity, lipid solubility, and crystal size of formazans) could be predicted from structural parameters. Possible applications of these correlations are discussed: to increase our understanding of existing staining systems and to guide the design and selection of new tetrazolium salts for histochemical use.

Formazans↗
PubMed1982

Estimating the effect of etching agents on plastic sections.

The effects on epoxy embedding media of certain etching procedures have been studied in two ways. Firstly the resin present in thick sections, before and after etching, was demonstrated by staining the plastic with Sudan Black B. Secondly the change in weight of plastic blocks after their exposure to etching procedures was measured. Methods involving halogens in acetone, alkoxides in ethanol or in methanol-benzene, and performic acid resulted in gross removal of plastic. Additionally, in keeping with the widespread belief that etching may increase permeability of plastic sections to stains, most of the etch methods studied, including organic solvents alone, did remove some plastic from the blocks.

Epoxy Resins↗
PubMed1981

Structure-staining relationships in histochemistry and biological staining. Part 3. Some comments on the intentional and artifactual staining of lipids.

The choice, basis and calculation of certain numerical coefficients for describing chemical structures of such staining reagents as dyes, enzyme substrates and visualising agents as described. The general value of electric charge, conjugated bond number, Hansch pi values, and molecular or ionic weights is emphasised. Hansch pi values as indicators of hydrophobic character, are then used to analyse reagents giving rise to the staining of neutral lipids. Dyestuffs used to stain lipids all have high Hansch pi values (greater than or equal to +1.0), with superior stains, e.g. Sudan Black B and Oil Red O, having values of greater than or equal to +7.0. In keeping with this, conversion of the basic dyes Nile Blue and Brilliant Cresyl Blue, with Hansch pi values of -2.4 and -3.6, into their oxazone derivatives, with Hansch pi values of +4.4, and 3.6, generates lipid staining compounds. Also in keeping with this correlation of lipophilia with Hansch pi values greater than +1.0 is the occurrence of artifactual lipid staining in enzyme histochemistry. Such artifacts can arise for instance when using certain naphthyl substrates which give rise to naphthoic intermediate reaction products, or when tetrazolium salts as used as visualising agents, yielding formazans as final reaction products. The Hansch pi values of the naphthols and formazans generated histochemically typically fall into the lipophilic range. Another artifact of lipid staining is the staining of basophilic entities, such as cell nuclei, by fat stains which carry amino substituents. Calculation of the Hansch pi values for protonated (and hence cationic) derivatives of such dyes yields values typical of basic dyes.

Animals↗
PubMed1980

The chemical nature of de Tomasi Schiff reagent. Changes of certain physical-chemical and histochemical properties during ageing.

Samples of de Tomasi's Schiff reagent were allowed to age. During this process physical, chemical and staining properties of the reagent were monitored. Certain physical and chemical properties of Schiff's reagent correlated with staining intensities. Thus a fall in the iodine titre and a rise in the pH (indicating loss of sulphur dioxide) accompanied falls of intensity of Feulgen-stained frog red-cell nuclei and of P.A.S.-stained cellulose sheet. Ageing sulphite solutions exhibited analogous physical and chemical changes. Thin layer and ion-exchange chromatography of Schiff's reagent demonstrated at lest four colourless (but colour generating) derivatives, all of which were cationic. NMR spectroscopy showed that the carbon skeletons of these compounds were simple and symmetrical. The ageing of Schiff's reagent appears to be primarily due to changes in its inorganic components. The aldehyde-reactive compounds probably differ only in the nature of the oxysulphur substituent on their central carbon atom.

Animals↗
PubMed1980

Structure-staining relationships in histochemistry and biological staining. I. Theoretical background and a general account of correlation of histochemical staining with the chemical structure of the reagents used.

A description is given of the choice, rationale and calculation of several numerical coefficients for describing the chemical structures of staining reagents such as dyestuffs, enzyme substrates, diazonium and tetrazolium salts. Using such coefficients, structure-staining correlations may be made for many staining systems. Examples include the structural correlations of the quantitative affinity measurements of the staining of chromatin with basic dyes, the tetrazolium salts for use as visualizing agents in enzyme histochemistry, and the use of bis-azo compounds as acid or basic dyes, or as fat stains. The value of such structure-staining correlation models for assisting the rational selection and design of new histochemical reagents, and for understanding old reagents and procedures, is discussed.

Chemical Phenomena↗
PubMed1980

Structure-staining relationships in histochemistry and biological staining. II. Mechanistic and practical aspects of the staining of elastic fibres.

Correlations between the structural features of dyes and staining performance for elastic fibres were investigated. Dyes studied included the traditional stains (such as Gomori's Aldehyde-Fuchsin and Weigert's Resorcin-Fuchsin), acid dyes used from alkaline aqueous-organic solvent mixtures (the Horobin-James system), and basic dyes used from acidic aqueous-ethanolic mixtures (the Taenzer-Unna system). In all three classes effective elastic fibre stains had large conjugated bond numbers, and were often hydrophobic (i.e. had high Hansch pi values). By choosing dyes with conjugated bond numbers at or over a critical value (25 for the TU system, 35 for the HJ) it is possible to select new and effective dyes for use in the HJ and TU staining systems. Mechanistically these results support the view that for typical commercial dyes and also for the traditional stains van der Waals attractions provide the important contributions to dye-elastic fibre affinities, with hydrophobic bonding playing a subsidiary role. However, supporting the views of Lillie, it was also noted that even hydrophilic dyes of low conjugated bond number could stain elastic fibres, if the dye carried a sufficiently reactive primary amino group as a substituent. The additional substituent groupings needed to generate such reactivity have been specified, for both acidic and alkaline reaction conditions.

Animals↗