Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.     

Fluorescent heterochromatin staining in primate chromosomes

Recently, in addition to quinacrine staining, fluorochrome techniques have been developed which brilliantly stain other heterochromatic regions. Two of these staining techniques are Distamycin/DAPI (DA/DAPI) and D287/170. We stained the chromosomes of all species of great apes and 14 species of primates (48 individuals) using these three fluorochrome techniques. Only african apes and man show brilliant quinacrine staining while, man and all the great apes show brilliant DA/DAPI staining and only species belonging to the hominoidea (including the siamang) showed bright D287/170 staining. In the lower primates a medium level of DA/DAPI fluorescence was found in some species with large amount of pericentromeric heterochromatin. Brilliant DA/DAPI staining could represent a derived trait linking all great apes and humans, while D287/170 may link all hominoidea. Fluorochrome staining is believed to be correlated with some satellite DNA sequences. However, data available on the chromosome location of satellite DNAs in non-human primates were derived from buoyant density fractions resulting in cross hybridization and now are not considered reliable. Before making any correlation between fluorochrome staining and satellite DNAs in non human primates there is need of data onin situ hybridization with cloned DNA sequences on primate chromosomes. These data would help clarify the evolution and relationship of satellite DNAs and heterochromatin in primates.     

Plasticity of repetitive DNA in response to metal stress in Bryophytes

Abstract

The relationship between environmental stresses and the genome was investigated by examining the behaviour of repetitive DNA in response to lead or cadmium in two Bryophytes, differing from a physiological and an ecological point of view, namely the aquatic moss L. riparium and the terricolous moss F. hygrometrica. Using different experimental approaches, a direct relationship was shown to exist in these two mosses between the metal-induced stress and repetitive DNA. In fact, in both organisms, metal treatment was accompanied by a selective amplification of some GC-rich repetitive DNA sequences forming peculiar agglomerates inside the nucleus; this amplification is quantitatively proportional to the time of exposure of the plants to the metals and stops upon removal of the metal from the culture medium. Results show that ribosomal DNA sequences are involved in this metal-induced repetitive DNA agglomerate formation, although they are not the only repetitive sequences present within the heterochromatic DNA agglomerates. The plasticity of the genome of the Bryophytes in response to external stimuli, and the fact that repetitive DNA is involved in this plasticity are discussed.     

Is repetitive DNA involved in male gametogenesis ofNarcissus papyraceus?

Summary The male gametophyte of the Spermatophyta is of particular interest when studying quantitative modifications of repetitive DNA which accompany plant differentiation. In fact, this well documented system offers the opportunity to investigate the phenomenon at the nuclear level. In particular, it permits comparison between single haploid nuclei which, though derived from the same mitotic division, have different, well-defined morphological and functional processes. The aim of the present work was to investigate the behaviour of repetitive DNA during male gametogenesis inNarcissus papyraceus. By the use of A+T and G+C specific fluorochromes (DAPI and Chromomycin A₃), and by in situ DNA digestion with appropriate restriction endonucleases (Eco RI and Hae III), it is shown that during the course of gametogenesis, generative and vegetative nuclei behave differently with respect to particular sections of the genome. Characteristic agglomerations of specific A+T rich DNA sequences can be observed, localized by the two distal ends of the spindle-shaped generative nucleus. The surface area occupied by the agglomerations differs significantly at the ends in all observed cases. The above agglomerations are present in most of the generative nuclei and are probably composed of repetitive DNA sequences. Some embryological questions are discussed in the light of these results.Abbreviations DAPI 4,6-diamidino-2-phenylindole - Eco RI Eco RI with star activity (N/AATTN) Dedicated to Prof. E. Battaglia on the occasion of his 75th birthday     

Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Verhalten tierischer Zellen in der Gewebekultur gegenüber einem basisch substituierten Benzimidazolderivat mit fluorochromierenden Eigenschaften

Zusammenfassung 33258 Hoechst, ein basisch substituiertes Benzimidazolderivat, beeinflußt Mitosen und Interphasezellen permanenter Zellinien in der Gewebekultur. Es kommt zu Mitosestörungen in der Anaphase und Telophase durch Verklumpen von Chromosomen, zu Zellvergrößerungen und zur Wachstumshemmung. Diese reversiblen und irreversiblen Veränderungen beruhen auf Verbindungen der Substanz mit der DNS der Zellkerne. Weiter wird über die Verwendung der Substanz als Fluorochrom zur Analyse von DNS-Schädigungen in Interphasezellen und an Mitosechromosomen berichtet.

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On the influence of a benzimidazol derivative (fluorochrome) on cell lines in tissue culture

Summary 33258 Hoechst, a benzimidazol derivative influences mitotic and interphase cells of permanent cell lines in tissue culture. Mitotic anomalities observed during anaphase and telophase are clumping of chromosomes, cell enlargement and inhibition of growth. These reversible and irreversible changes are due to the reaction between the substance and the DNA in the nucleus. Further is reported about the utilization of the substance as fluorochrome to analyse DNA damage in interphase cells and mitotic chromosomes.

     

Detection of endogenous and immuno-bound peroxidase — The status Quo in histochemistry

The discovery of synthetic dyes goes back to 1856 and launched the development of the whole chemical and pharmaceutical industry. In life sciences synthetic dyes represent indispensable tools for the microscopic and macroscopic level. Small dyes have the advantage of their easy adaptability to various measuring equipments. By way of structural modification of the chromophore portion, dye labels can be tailored that they absorb and emit light at desired wavelengths ranging from the UV to the near infrared region of the spectrum. Assisted by the development of light measuring techniques and the commercial availability of highly sensitive equipment, today luminescent labels represent most sensitive detection tools in life sciences and dominate over chromogen based techniques. However, for detection of active sites of peroxidase (PO) so far fluorescent labels have been confined to only a few substrates while a broad variety of well-established chromogenic techniques exist. This review covers fluorescent and chromogenic approaches for the permanent detection of immuno-bound and endogenous PO-activity in fixed cells and tissues. Thereby the tailoring of suitable dye labels is additionally challenged by two demands: (1) The applied dye (or its precursor) must act as enzyme substrate specifically and (2) the enzymatic impact must furnish an insoluble dye product from easy soluble starting materials in a very quick reaction. Hence it is not surprising that among PO-substrates (and enzyme substrates generally), dye conjugates represent only an exception while most of these labels represent reactive dyes or suitable precursors. Chromogenic and fluorescent approaches for the permanent labeling of enzymatic sites are compiled. Furthermore, various area-spanning PO-detection principles are discussed ranging from transmission light (TLM) and fluorescence light (FLM) microscopy (chromogenes, flourochromes, fluorescent chromogenes, chromogenes with nonlinear optical properties) to correlated transmission electron microscopy (TEM; photoconversion of specific chromogenic reaction products, electron opaque and/or osmiophilic chromogenic substrates). Also, approaches for reflectance laser microscopy (RLM), polarization microscopy (PM), and correlative TLM, FLM, and multiphoton fluorescence microscopy (MFM) are discussed.     

Effects of lead on the nuclear repetitive DNA of the mossFunaria hygrometrica (Bryophyta)

Summary Funaria hygrometrica gametophytes were grown in in vitro controlled systems in the presence and absence of lead. Their nuclear genomes were then analyzed directly in situ with A+T and G+C specific fluorochromes, image analysis and statistical data elaboration by specific software, in order to characterize the different fractions of repetitive DNA. The results reveal qualitative and quantitative differences between the nuclear genomes of lead-stressed and unstressed individuals. These differences seem to consist of a significant increase in G+C rich repetitive DNA sequences in nuclei of the stressed individuals. These sequences form well defined agglomerates, generally situated adjacent to the nucleolar region, which increase in both size and number in the presence of lead. Some hypotheses are discussed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - BBM bold basal medium     

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Mitochondrial membrane permeabilization (MMP) is considered as the “point-of-no-return” in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential (ΔΨ_m). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several techniques to measure MMP by cytofluorometry and fluorescence microscopy have been developed. Here, we summarize the currently available methods for the detection of MMP, and provide a comparative analysis of these techniques.     

Classification and naming of dyes,stains and fluorochromes

A classification of dyes and other colorants is proposed, based on the chemical features responsible for their visibility and generally consonant with the writings of modern color chemists. The scheme differs in several respects from that of the Colour Index (CI), but it retains some traditional small groups of dyes that include biological stains. Natural dyes, recognized as a group in the CI, are placed with or near synthetic dyes with identical or similar chromophores. The new scheme also provides categories for dyes and fluorochromes that do not have places in the CI classification. Some CI categories, including lactones, aminoketones and hydroxyketones, are not recognized in this new scheme, which is adopted in the forthcoming 10th edition of Conn's Biological Stains: a Handbook of Dyes and Fluorochromes for Use in Biology and Medicine. Some rules are also set out for the spelling of trivial names, which has long been inconsistent in scientific literature. The ending '-ine' is used for compounds derived from organic bases (e.g., fuchsine and thionine, not fuchsin or thionin), and names ending in '-in' are for compounds that are not bases or their derivatives (e.g., eosin and phloxin, not eosine or phloxine). Initial capital letters are used only for words that are names of people or places (e.g., Nile blue or Congo red) and for the 'generic' components of CI application names (as in Acid yellow 36). Other words, including trade names that have fallen into common usage are not capitalized (e.g., alcian blue, biebrich scarlet, coomassie blue). The recommended spellings of some dyes differ from those commonly seen in vendors' catalogs and in biological publications, but they are generally consistent with English and American dictionaries, with recent writings in English by color chemists, and with the trivial names of other organic compounds.