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R W Horobin

Publications and source records attributed to R W Horobin.

At least 19 recordsLinked to original sources
PubMed1994

Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents.

The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.

Animals↗
PubMed1994

Overview of Microwave Applications in the Neurosciences.

This paper gives an overview of Microwave Applications in the Neurosciences and introduces this issue of the Journal of Neuroscience Methods. Emphasis is laid on: temperature, hotspots, hazards and cycle times in the use of the microwave oven. Consequences of the tissue dimensions and masking of antigens are discussed. Special problems encountered are: bulk preparation of specimens, brain electron microscopy, cooling of specimens during microwaving and the combination of microwave techniques. The recent developments discussed are vacuum-microwave application for large brain specimens and antigen retrieval.

Animals↗
PubMed1993

Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity.

The reduction of four tetrazolium cations (TCs), nitroblue tetrazolium (NBT), neotetrazolium (NT), methylthiazolyldiphenyltetrazolium (MTT) and iodonitrophenyltetrazolium (INT), by viable micro-organisms, immobilized on glass cover slips, was examined by light microscopy with a view to determining a systematic basis for applying these reagents as cytochemical indicators of microbial viability and activity. The potential value of histochemical information about TC reactions for developing their microbiological applications was also assessed. INT and MTT detected viable cells more readily than NBT and NT. In order to obtain cell-localized formazan, MTT required cobalt ions in the reaction mixture and INT reactions had to be assessed soon after mounting. In general, formazan deposition could be accelerated by the addition of glucose and an intermediate electron carrier (IEC) to the reaction mixture, although inhibitory effects of IECs were also detected. Cultures in exponential phase, in stationary phase and inhibited by chloramphenicol could be differentiated with MTT but not with INT. For some organisms, notably Candida albicans, Pseudomonas aeruginosa and Enterococcus faecalis. TC reactions proved to be a relatively insensitive means of demonstrating viability. Two parameters used in selecting TCs for histochemical reactions, lipophilicity and reducibility appeared to be predictive for the relative sensitivity of these reagents as indicators of cell viability. The concepts of substantivity, a measure of non-specific interactions between reagents and staining substrates, and TC oxygen sensitivity, the effect of competition between oxygen and TCs for electrons, were found to be relevant to formazan deposition in live microbes. These findings support the use of TCs as cytochemical probes of microbial activity in defined settings and the use of histochemical knowledge to support further development of these techniques.

Bacteria↗
PubMed1992

The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue.

The dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration. The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.

Animals↗
PubMed1992

Staining sections of water-miscible resins. 2. Effects of staining-reagent lipophilicity on the staining of glycol-methacrylate-embedded tissues.

Glycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty-seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol-water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P greater than 2. In keeping with this, use of GMA semi-permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains--in terms of the probable occurrence of high resin background staining and low tissue sensitivity--is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P less than 0; molecular weight less than 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P greater than 2; 550 less than ionic weight less than 1000 Da), give strong background staining.

Animals↗
PubMed1991

Predicting the behaviour and selectivity of fluorescent probes for lysosomes and related structures by means of structure-activity models.

Cultured rat fibroblasts were exposed to 50 fluorescent probes of varied physicochemical characteristics. Probe concentrations, fluorochrome excitation wavelength and period of illumination, and cell-probe contact time were varied. Structure-activity relationships defining a number of classes of fluorescent probes for lysosomes and related processes and compartments were demonstrated. Numerical specifications are now available for several familiar classes of probes: (a) acidotropic weak bases, used as markers for low pH compartments; (b) markers of adsorptive pinocytosis, involving non-specific protein binding; (c) markers for fluid phase pinocytosis; and (d) viability stains involving intralysosomal enzymic activity. Two novel classes of probes have also been specified numerically: (a) acid-precipitated weak acids, as markers for low pH compartments; and (b) lipid-binding markers of adsorptive pinocytosis. Overall, these structure-activity models provide a tool for predicting whether or not compounds enter cells; and whether they accumulate in lysosomes and related compartments. Pathways of entry are also predicted. This tool should permit design and selection of improved probes, and provide a better understanding of existing reagents. Moreover these models are expected to be applicable to interactions between any non-polymeric xenobiotic with lysosomes and related compartments.

Animals↗
PubMed1991

Accumulation of fluorescent non-cationic probes in mitochondria of cultured cells: observations, a proposed mechanism, and some implications.

Cultured rat fibroblasts were exposed to millimolar concentrations of forty-four non-cationic fluorescent probes, of very varied physico-chemical properties. Mitochondrial staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non-ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge less than or equal to 0; log P (less-ionized form) less than 0; and pKa approximately 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion-trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here.

Animals↗
PubMed1990

Interaction of molecular probes with living cells and tissues. Part 2. A structure-activity analysis of mitochondrial staining by cationic probes, and a discussion of the synergistic nature of image-based and biochemical approaches.

Cultured rat fibroblasts were exposed to 41 cationic fluorescent probes of very varied hydrophilicity/lipophilicity. Outcome of probe-cell interaction fell into one of the following categories: probe failed to enter the cells; probe accumulated on cell surfaces; probe accumulated in mitochondria, and/or in other intracellular regions. The observations were analysed using a Simplistic Chinese Box (SCB) approach, and the following conclusions were reached. It was the hydrophilic probes which failed to enter cells, whilst extremely lipophilic probes were retained on the cell surfaces. Only the slightly lipophilic cationic probes were permeant, and accumulated in mitochondria. Using the probes log P values to model hydrophilicity/lipophilicity, effective cationic mitochondrial stains can be specified numerically so: 0 less than log P probe less than +5. This SCB model was used to rationalise a variety of earlier observations on the action of mitochondrial probes. The applicability of the SCB approach to integrate image-based and biochemical investigations was demonstrated by using the action of chlorpromazine on mitochondrial action as a case example.

Animals↗
PubMed1990

A numerical procedure for choosing effective, low toxicity plasticizers for glycol methacrylate embedding.

A numerical method for selecting low toxicity plasticizers for glycol methacrylate (GMA) embedding mixtures is described. It involves use of various numerical parameters, namely; molecular weight, melting point, solubility parameter, and toxicity data. On the basis of these parameters, nine plasticizers were selected. Their effects on microtomy and tissue processing, and also their influence on tissue morphology and staining, were investigated. For tissue processed at low temperatures into GMA, using ethanol dehydration, the following compounds were as satisfactory as 2-butoxyethanol whilst being less toxic: 2-isopropoxyethanol, 2-(2-methoxyethoxy)ethanol, 2-(2-ethoxyethoxy)ethanol, 2-(n-butoxyethoxy)ethanol. However for tissues processed using the plasticizer as dehydrating agent, the optimum plasticizers were 2-isopropoxyethanol, 2-(2-methoxyethoxy)ethanol and 2-(2-ethoxyethoxy)ethanol. It is possible to give a numerical specification of the preferred plasticizers, and for the first procedure this is: a solubility parameter in the range 21-26 J0.5 cm-1.5 or higher, a melting point well below 0 degree C, and a rat oral LD50 greater than 12.52 mmol kg-1. It was also possible to analyse the differential effects of the dehydrating agents on histochemical and enzyme histochemical staining on a numerical basis, using a structure-activity relations approach.

Animals↗
PubMed1990

Interactions of molecular probes with living cells and tissues. Part 1. Some general mechanistic proposals, making use of a simplistic Chinese box model.

A simple and generalised model-termed the simplistic Chinese box [SCB] model-for the interaction of molecular probes with living systems is described. The SCB model includes the following assumptions. That living systems may be considered as built from biologically defined boxes, e.g. whole cell, nucleus, nucleoli. That movement of molecular probes into and through these boxes is strongly influenced by box wall permeability, which in turn is largely dependent on the simple physicochemical properties of probe and wall. That retention of probes in boxes is influenced by permeability of the walls and by trapping of probes by boxes and walls, the latter effect also being strongly dependent on simple physicochemical properties. That important physicochemical properties include electric charge, hydrophilicity/lipophilicity, non-specific protein binding, and molecular size. That, since all these factors can be expressed or modelled numerically, SAR methodology is an appropriate technique for analysing molecular probe investigations.

Biological Transport↗
PubMed1990

Staining sections of water-miscible resins. 1. Effects of the molecular size of stain, and of resin cross-linking, on the staining of glycol methacrylate embedded tissues.

Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. 'Large' dyes (greater than 1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. 'Small' dyes (less than 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dye of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.

Animals↗
PubMed1989

Understanding Romanowsky staining. 2. The staining mechanism of suspension-fixed cells, including influences of specimen morphology on the Romanowsky-Giemsa effect.

Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.

Azure Stains↗
PubMed1989

Use of tissue-free glycol methacrylate sections as semi-permeable membranes: a simple way to shorten incubation times and to improve localization in enzyme histochemistry.

Placing 2-microns sections of tissue-free glycol methacrylate on top of tissue sections is a simple way of forming semipermeable membranes to enhance enzyme histochemical staining. For demonstrating alkaline phosphatase in glycol methacrylate-embedded kidney by a standard azo dye method, such membranes enabled incubation times to be reduced to 1-2 hr, with azo dye reaction product being more crisply localized as compared to sections stained without membranes. Such effects are possible because the membranes are highly permeable to small molecules (e.g., substrate and diazonium salt), slightly permeable to molecules of moderate size (e.g., the final reaction product), and impermeable to large molecules (e.g., alkaline phosphatase and other tissue biopolymers). The implications of these findings for enzyme histochemistry and for enzyme-labeled antibody staining are discussed.

Acrylates↗
PubMed1989

Preparation and characterization of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and related stilbene disulfonates.

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and other 4,4'-stilbene-2,2'-disulfonate derivatives used as reagents in histochemistry and physiology have been prepared in their E isomeric form, and rearranged to the Z isomers by irradiation with visible light. Infrared, and 1H and 13C nuclear magnetic resonance spectra were recorded for these compounds, and used to establish the chemical structures. In particular, it was shown that the E-isomer of SITS decomposed in aqueous solution by hydrolysis of both the acetamido and isocyano groups yielding a diamine; disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also decomposed in solution, while disodium 4,4'-dinitrostilbene-2,2'-sulfonate (DNDS) rearranged from the E-isomer to the Z-isomer when solutions were kept unprotected from light. These results indicate that benchworkers should not be surprised when commercial samples of such stilbenes contain large amounts of various types of impurities.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

One-bath trichrome staining: investigation of a general mechanism based on a structure-staining correlation analysis.

Dye pairs of contrasting colours were selected from acid dyes of varied chemical characteristics. The 44 dye pairs were investigated in a one-bath trichrome staining system in which the dye-baths were strongly acid. Dye concentrations, concentration ratios and staining times were varied for each dye pair. Thirty dye pairs stained collagen fibres distinctly different colours to muscle cytoplasm, while 14 dye pairs gave muddy, non-selective staining. Comparison of dye structures showed that in selective pairs the larger dye always stained the collagen fibres, with cytoplasm being coloured by the smaller species. With 28/30 of the selective dye pairs the differences in anionic weights of the dyes was greater than 200. However, in dye pairs giving non-selective staining, the anionic weights of the members of 13/14 of the dye pairs differed by less than 200. As no other structural feature correlated so clearly with selectivity, it was concluded that the selectivity of one-bath trichromes is diffusion-rate controlled, involving the interaction of differentially permeable tissue sites (collagen being more permeable than muscle cytoplasm) with dyes diffusing at different rates (large dyes slower than small). In keeping with this, lengthening staining times reduced staining selectivity. The rate control mechanism suggested a rational trouble-shooting guide for one-bath trichromes, encompassing such practical factors as dye concentration, embedding medium, fixative, dye-bath pH, section thickness and staining time.

Animals↗
PubMed1988

Chemical structures and staining mechanisms of Weigert's resorcin-fuchsin and related elastic fiber stains.

Chemical properties of Weigert's resorcin-fuchsin, orcinol-new fuchsin, Sheridan's crystal violet and their resorcinol-free analogues were investigated using reverse-phase and gel filtration chromatography, electrophoresis, and visible light spectroscopy. Their staining properties were also studied. It was concluded that 1) the staining components of Weigert's resorcin-fuchsin, orcinol-new fuchsin and their resorcinol-free analogues are all indamine oligomers, 2) resorcinol is required for the production of Sheridan's crystal violet, the staining components of which consist of crystal violet substituted by varying numbers of resorcinyl substituents, 3) the staining components of all preparations are cationic (i.e., basic) dyes, 4) iron is present in staining solutions as the tetrachloroferrate anion (FeCl4-) and not as Fe or as a dye-chelate, and 5) since even the smallest Weigert's resorcin-fuchsin, orcinol-new fuchsin or Sheridan's crystal violet component has a conjugated bond number of 32, the observed staining of elastic fibers is only as expected.

Animals↗