Characterization of six textile dyes as fluorescent stains for flow cytometry.

Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.     

Standardization of biological dyes and stains: pitfalls and possibilities.

The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.     

Standardization of biological dyes and stains: pitfalls and possibilities

Summary The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.This paper is dedicated to my academic teacher, Prof. Dr. D.H. Wittekind, on the occasion of his 70th birthday     

Karyokinetic reactions in rat kidney proximal convoluted segment epithelium on long-term lead supply with drinking water

In the rat kidney proximal convoluted segment epithelium the development of intranuclear inclusion bodies was observed after long-term lead administration with the drinking water. The inclusion bodies were PAS-positive. By electron microscopy they were identified as composed of filaments and granules lacking any kind of surrounding membrane. During the experiment, the nuclear volumes increased commensurately with the duration of lead exposure. The nuclear volume increase was considered a physiological reaction since it began long before the first appearance of intranuclear inclusion bodies.     

Intranuclear inclusions in urinary cytology

Twenty-one urinary cytology specimens from 16 patients showed red intranuclear inclusion bodies. The literature pertinent to these inclusions mainly suggests that lead poisoning is a cause for such inclusions. Histories were reviewed for possible causes, particularly lead poisoning, associated medications, associated illness or relationship to neoplasia. The only consistent associations found were that all the patients were women over the age of 50 and that all inclusions were found in voided urine specimens. Four slides containing inclusions were stained by acid-fast methods, with negative results. The origin of these inclusions remains unknown.     

Indole reactions of mast cells and enterochromaffin cells related to fixations with and without aldehydes

Summary Survey of a considerable number of rat, mouse and hog tissues which presented large numbers of mast cells in preparations stained with toluidine blue and other metachromatic or basic dyes at low pH levels, revealed numbers of oval bodies of about the same size as mast cells which reacted weakly or even moderately to the postcoupled benzylidene indole reaction. The numbers of these were always less than that of mast cells in toluidine blue sections of the same blocks. They never occurred in clusters of perhaps 15–20 in a single high power field, as mast cells often do. Smooth and especially striated muscle which often formed the background tissue where most mast cells are found with metachromatic stains, regularly present indole reactions due to protein tryptophan. This is usually equal to or stronger than that in the supposed mast cells.Indole reactive bodies whose morphology suggests mast cells are also present in similar numbers in formaldehyde and glutaraldehyde fixed tissue as well as with aldehyde free fixations. Glutaraldehyde and formaldehyde are known to inhibit the benzylidene reaction of 5-HT in vitro (30 min for glutaraldehyde, 3 h for formaldehyde) (Lillie, 1977). This action was avoided in mercury and lead heavy metal fixations and in acetone, Carnoy, chloroform methanol and similar fixations.The mast cell-like bodies are best explained as tangential or oblique sections of individual muscle fibers. We have described the same phenomenon with the ferric ferricyanide (Golodetz-Unna, 1909) reaction (Lillie et al., 1978a), and the PCB reaction is that of tryptophan in these muscle cell sections.In contrast to the DMAB type reaction failure acid diazosafranin successfully demonstrated mast cells with both aldehyde and aldehyde free fixations. This reaction has been shown to occur with 5-HT and 5-HTP (Lillie et al., 1973).     

Improving Biological Dyes and Stains: Quality Testing Versus Standardization

This paper discusses the impact of both standardization and quality testing of dyes and stains in biology and medicine. After a brief review of why standardized dyes and stains are not presently available commercially, two types of testing and ways of improving dye quality are described. National or international organizations could be established to define standardization of dyes and stains. Standardization would be specifically defined as a list of physico-chemical parameters such as elaborated in this paper. Commercial batches of comparable quality may be labeled by the supplier as “standard dye.” a procedure currently performed by the European Council for Clinical and Laboratory Standardization (ECCLS). Also recommended to improve dye quality is commercial dye testing by independent laboratories with subsequent certification for use. This sort of quality control is currently carried out in the United States by the Biological Stain Commission (BSC). The advantages and disadvantages of both techniques and the use of image analysis for the definition of standards are discussed. A combination of both the BSC testing protocols and the ECCLS standards should be established for extended quality control of biological dyes and stains.     

Chronic lead intoxication causes a brain-specific nuclear protein to accumulate in the nuclei of cells lining kidney tubules

A characteristic feature of chronic lead intoxication is the induction of intranuclear inclusion bodies in cells lining kidney proximal tubules. These are relatively insoluble lead- and protein-rich structures which may serve a protective function by sequestering lead. The most abundant protein of isolated inclusion bodies, p32/6.3, has been partially characterized by use of a monoclonal antibody. As predicted by biochemical analysis, p32/6.3 occurs in kidney only in conjunction with lead intoxication and inclusion body formation. It does not accumulate in other tissues as a result of lead exposure. Unexpectedly, p32/6.3 was found to be a constitutive protein of adult brain, occurring primarily in the cerebral cortex. Within this tissue, both neurons and astrocytes contained p32/6.3. The brain p32/6.3 was concentrated in the insoluble nuclear protein or matrix fraction, a localization reminiscent of the intranuclear inclusion bodies from lead-exposed kidney. Brain p32/6.3 was detected in rat, mouse, dog, man, and chicken. In rat brain, the appearance of p32/6.3 was developmentally regulated. Only traces were detected 3 days after birth but within 1-2 weeks adult levels were achieved. The presence in brain of a protein which is involved in a potentially protective response to lead suggests that the brain may also have such a protective mechanism.     

New amino and acetamido monomethine cyanine dyes for the detection of DNA in agarose gels

Some new monomethine cyanine dyes derived from quinoline and benzothiazole have been prepared and characterized by (1)H and (13)C NMR, FTIR, FABHRMS, and visible spectroscopy. The dyes containing amino and acetamido groups were conveniently synthesized by the condensation of two p-toluenesulfonate heterocyclic quaternary salts and were obtained in the forms of iodide, bromide, and tosylate counteranions. These dyes were compared to ethidium bromide as stains for DNA in electrophoretic gels. The overall results obtained for the sensitivity of these dyes suggest the suitability of acetamido moiety over the amine one and bromide as the counteranion when compared with iodide and tosylate, with a similar capacity of DNA detection in relation to the ethidium bromide stain over the concentration range of 1-3ng.     

Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells

The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.     

Probable herpesvirus infection in an eastern cottontail (Sylvilagus floridanus).

One wild eastern cottontail (Sylvilagus floridanus) from Milwaukee County, Wisconsin was necropsied. The lungs contained numerous multifocal, circumscribed, tan foci; the spleen was markedly enlarged and had a mottled reddish tan color; and the brain had a red to tan friable tract in the left hemisphere. Microscopically, the lung had a severe bronchiolitis and pneumonia. The bronchiolitis was characterized by epithelial cells containing eosinophilic intranuclear inclusion bodies. The encephalomalacia of the left cerebral cortex featured tissue disruption and astrocytes or neurons containing intranuclear inclusion bodies. Herpesvirus particles were found within the bronchiolar epithelial cells. Based on histopathological and ultrastructural findings, a herpesvirus seemed the most likely etiologic agent.     

High-throughput screens for fluorescent dye discovery

A recent screen of a combinatorial library of fluorescent compounds discovered fluorescent dyes that were able to distinguish myoblasts from differentiated myotubes. New fluorescent dyes that respond to biologically relevant changes in cell state or type are useful as stains in a wide variety of biological experiments, including high-throughput screens for chemical and genetic regulators. Combining this approach with microscopy imaging is likely to be even more powerful and might lead to the discovery of new dyes with interesting and useful properties.     

Further morphological and histochemical studies on the yolk nucleus and associated cell components in the developing oocyte of the Indian wall lizard

In March through April when the oocyte growth in the ovaries of the wall lizard (Hemidactylus) is very rapid, the yolk nucleus continues to persist through various stages of previtellogenesis. This persisting yolk nucleus and associated cell components have been studied with histochemical techniques. The spherical and dense yolk nucleus stains for protein, lipoprotein and RNA. It does not form any close morphological association with the other cell components such as the mitochondria, lipid bodies (L₂), spaces or canals, diffuse sudanophilic substance and dense bodies, which are arranged into three zones round the yolk nucleus proper. The mitochondria stain for lipoprotein; the L₂ bodies consist of phospholipid; the spaces do not contain any material demonstrable with histochemical techniques; and the ooplasm containing the diffuse sudanophilic substance and dense bodies shows lipoprotein, protein and RNA. Eventually, the yolk nucleus disintegrates, and its substance as well as the other cell components are distributed in the cortical ooplasm of oocytes which are ready to form the yolk bodies. Concepts of the origin, morphology, cytochemistry and function of the yolk nucleus in the oocytes of invertebrates and vertebrates, which have come about recently through the application of cytochemical and submicroscopical techniques, are discussed.     

Negative Stains in the Demonstration of Bacteria

Since certain objectionable features have been observed in the old technics of preparing negatively stained bacteria slides, modifications of these methods have been attempted, and studies have been made of various dyes not heretofore described as negative stains. India ink, nigrosin, indulin, Congo red, Poirier's blue, anilin blue (methyl blue), China blue, blue de Lyon, blue de Lyon O, and night blue are recommended. Dyes of the same name, but sold by different companies, often present different effects.     

Intranuclear dynamics of the Nup107-160 complex

Nup98 is a glycine-leucine-phenylalanine-glycine (GLFG) repeat–containing nucleoporin that, in addition to nuclear transport, contributes to multiple aspects of gene regulation. Previous studies revealed its dynamic localization within intranuclear structures known as GLFG bodies. Here we show that the mammalian Nup107-160 complex (Y-complex), a major scaffold module of the nuclear pore, together with its partner Elys, colocalizes with Nup98 in GLFG bodies. The frequency and size of GLFG bodies vary among HeLa sublines, and we find that an increased level of Nup98 is associated with the presence of bodies. Recruitment of the Y-complex and Elys into GLFG bodies requires the C-terminal domain of Nup98. During cell division, Y-Nup–containing GLFG bodies are disassembled in mitotic prophase, significantly ahead of nuclear pore disassembly. FRAP studies revealed that, unlike at nuclear pores, the Y-complex shuttles into and out of GLFG bodies. Finally, we show that within the nucleoplasm, a fraction of Nup107, a key component of the Y-complex, displays reduced mobility, suggesting interaction with other nuclear components. Together our data uncover a previously neglected intranuclear pool of the Y-complex that may underscore a yet-uncharacterized function of these nucleoporins inside the nucleus, even in cells that contain no detectable GLFG bodies.     

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.     

New stains for blood and bone marrow cells.

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

New Stains for Blood and Bone Marrow Cells

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an assymetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

Report from the President

Although the original Commission on Standardization of Biological Stains was first organized in 1921, it was not until 1944 that this was incorporated as an independent, nonprofit organization known as the Biological Stain Commission (see Clark 1974). The certification of dyes, as indicated by special labels purchased by manufacturers or vendors for attachment to the dye containers, originated with the parent organization and has continued to this day. The objectives of the Biological Stain Commission (BSC) are 1) to identify and standardize the content and performance of dyes and dye preparations used in staining biological tissues and products, 2) to issue labels of certification to companies that buy these to inform consumers that their certified dyes meet the specifications of the BSC, 3) to carry out and to support investigations on dyes and their performance, 4) to publish scientific data concerning biological stains and their use, and 5) to maintain, through scientific meetings and correspondence, an active “dialogue” among scientific and industrial personnel concerned with biological stains. The present report summarizes Commission activity and some of the changes that have occurred during the past five years.