Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

Isohematein as a Biological Stain

The possible use of isohematein as a biological stain is considered. Certain characteristics of the dye are discussed in relation to staining technic. A preliminary series of experiments is described. The stomach of the frog, skeletal muscle of the frog, and spinal cord of the cat were used as representative tissues. The dye has greater tinctorial power than hematoxylin (hematein) but it is not so selective for nuclei. The results at hand indicate that the dye may have some value as a differential stain for nerve cell bodies. Fibrillae in smooth muscle cells and cross striatums in skeletal muscle were also brought out.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled beta-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.     

DAPI: a DNA-Specific Fluorescent Probe

DAPI (4′,6-diamidino-2-phenylin-dole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.     

DAPI: a DMA-Specific Fluorescent Probe

DAPI (4',6-diamidino-2-phenylin-dole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Multiple biological activities of curcumin: a short review

Turmeric (Curcuma longa rhizomes), commonly used as a spice is well documented for its medicinal properties in Indian and Chinese systems of medicine. It has been widely used for the treatment of several diseases. Epidemiological observations, though inconclusive, are suggestive that turmeric consumption may reduce the risk of some form of cancers and render other protective biological effects in humans. These biological effects of turmeric have been attributed to its constituent curcumin that has been widely studied for its anti-inflammatory, anti-angiogenic, anti-oxidant, wound healing and anti-cancer effects. As a result of extensive epidemiological, clinical, and animal studies several molecular mechanisms are emerging that elucidate multiple biological effects of curcumin. This review summarizes the most interesting in vitro and in vivo studies on the biological effects of curcumin.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Recent Updates in Curcumin Pyrazole and Isoxazole Derivatives: Synthesis and Biological Application

Curcumin is an admired, plant‐derived compound that has been extensively investigated for diverse range of biological activities, but the use of this polyphenol is limited due to its instability. Chemical modifications in curcumin are reported to seize this limitation; such efforts are intensively performed to discover molecules with similar but improved stability and better properties. Focal points of these reviews are synthesis of stable pyrazole and isoxazole analogs of curcumin and application in various biological systems. This review aims to emphasize the latest evidence of curcumin pyrazole analogs as a privileged scaffold in medicinal chemistry. Manifold features of curcumin pyrazole analogs will be summarized herein, including the synthesis of novel curcumin pyrazole analogs and the evaluation of their biological properties. This review is expected to be a complete, trustworthy and critical review of the curcumin pyrazole analogs template to the medicinal chemistry community.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

Staining properties of aldehyde fuchsin analogs.

This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.     

Staining of macromolecules: possible mechanisms and examples

This review is based on a presentation given at the Biological Stain Commission meeting in June 2008. I discuss staining as an interaction between dye, solvent, and biological macromolecules. Most staining takes place in water, where the physico-chemical properties of the macromolecules are particularly important. Staining from aqueous solution is summarized. The first step is diffusion–ion exchange, which builds up the dye ion concentration close to the appropriately charged tissue constituents. While charge interactions are important for selectivity and build-up of dye ions around specific tissue and cell constituents, they have in most cases little to do with actual dye binding. The next step, actual binding, is predominantly between aromatic and other non-polar parts of the dye and corresponding groups in the tissue constituent. This results in a reduction of the total hydrophobic area exposed to water, hence the term hydrophobic interaction. Because dye binding is predominantly by dispersive forces, the larger the aromatic dye system and the fewer the number of charges on the dye, the greater the substantivity or affinity. Some relatively straightforward anionic or cationic one-step staining systems are discussed also. These include amyloid staining with Congo red, elastin staining with orceins, collagen staining with picrofuchsin, DNA–RNA staining with methyl green-pyronin Y, acid heteroglycan staining with Alcian blue, and metachromatic staining.     

Curcumin as a natural regulator of monocyte chemoattractant protein-1

Monocyte chemoattractant/chemotactic protein-1 (MCP-1), a member of the CC chemokine family, is one of the key chemokines that regulate migration and tissue infiltration of monocytes/macrophages. Its role in the pathophysiology of several inflammatory diseases has been widely recognized, thus making MCP-1 a possible target for anti-inflammatory treatments. Curcumin (diferuloylmethane) is a natural polyphenol derived from the rhizomes of Curcuma Longa L. (turmeric). Anti-inflammatory action underlies numerous pharmacological effects of curcumin in the control and prevention of several diseases. The purpose of this review is to evaluate the effects of curcumin on the regulation of MCP-1 as a key mediator of chemotaxis and inflammation, and the biological consequences thereof. In vitro studies have shown that curcumin can decrease MCP-1 production in various cell lines. Animal studies have also revealed that curcumin can attenuate MCP-1 expression and improve a range of inflammatory diseases through multiple molecular targets and mechanisms of action. There is limited data from human clinical trials showing the decreasing effect of curcumin on MCP-1 concentrations and improvement of the course of inflammatory diseases. Most of the in vitro and animal studies confirm that curcumin exert its MCP-1-lowering and anti-inflammatory effects by down-regulating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway. As yet, there is limited data from human clinical trials showing the effect of curcumin on MCP-1 levels and improvement of the course of inflammatory diseases. More evidence, especially from human studies, is needed to better assess the effects of curcumin on circulating MCP-1 in different human diseases and the role of this modulatory effect in the putative anti-inflammatory properties of curcumin.     

Curcumin as a wound healing agent

Turmeric (Curcuma longa) is a popular Indian spice that has been used for centuries in herbal medicines for the treatment of a variety of ailments such as rheumatism, diabetic ulcers, anorexia, cough and sinusitis. Curcumin (diferuloylmethane) is the main curcuminoid present in turmeric and responsible for its yellow color. Curcumin has been shown to possess significant anti-inflammatory, anti-oxidant, anti-carcinogenic, anti-mutagenic, anti-coagulant and anti-infective effects. Curcumin has also been shown to have significant wound healing properties. It acts on various stages of the natural wound healing process to hasten healing. This review summarizes and discusses recently published papers on the effects of curcumin on skin wound healing. The highlighted studies in the review provide evidence of the ability of curcumin to reduce the body's natural response to cutaneous wounds such as inflammation and oxidation. The recent literature on the wound healing properties of curcumin also provides evidence for its ability to enhance granulation tissue formation, collagen deposition, tissue remodeling and wound contraction. It has become evident that optimizing the topical application of curcumin through altering its formulation is essential to ensure the maximum therapeutical effects of curcumin on skin wounds.     

Modulation of apoptosis-related cell signalling pathways by curcumin as a strategy to inhibit tumor progression

A hallmark of cancer is resistance to apoptosis, with both the loss of proapoptotic signals and the gain of anti-apoptotic mechanisms contributing to tumorigenesis. As inducing apoptosis in malignant cells is one of the most challenging tasks regarding cancer, researchers increasingly focus on natural products to regulate apoptotic signaling pathways. Curcumin, a polyphenolic derivative of turmeric, is a natural compound derived from Curcuma longa, has attracted great interest in the research of cancer during the last half century. Extensive studies revealed that curcumin has chemopreventive properties, which are mainly due to its ability to arrest cell cycle and to induce apoptosis in cancer cells either alone or in combination with chemotherapeutic agents or radiation. The underlying action mechanisms of curcumin are diverse and has not been elucidated so far. By regulating multiple important cellular signalling pathways including NF-κB, TRAIL, PI3 K/Akt, JAK/STAT, Notch-1, JNK, etc., curcumin are known to activate cell death signals and induce apoptosis in pre-cancerous or cancer cells without affecting normal cells, thereby inhibiting tumor progression. Several phase I and phase II clinical trials indicate that curcumin is quite safe and may exhibit therapeutic efficacy. This article reviews the main effects of curcumin on the different apoptotic signaling pathways involved in curcumin induced apoptosis in cancer cells via cellular transduction pathways and provides an in depth assessment of its pharmacological activity in the management of tumor progression.     

Staining of microsporidian spores with diamidine phenylindole

A number of microscopic techniques and dyes are available to diagnose microsporidian infections in invertebrate and vertebrate hosts. Among these, DNA-specific fluorochrome DAPI is widely used to stain DNA in prokaryotic and eukaryotic cells, alone or in combination with other histochemical or fluorescent dyes. Moreover, this dye also binds to membraneous structures and protein complexes. In our studies, DAPI was used to stain spores of microsporidia infecting orthopteran, coleopteran, dipteran and lepidopteran insect hosts. DAPI staining of diplokarya helped to discriminate the Nosema-like microsporidian spores from spore-shaped bodies lacking this characteristic staining. It was found, moreover, that non-DNA staining occurred in many cases and other components of the spores were stained: the exospore, the cytoplasm, the extruded polar filament and the polaroplast. Staining of these structures was feeble as compared to DNA and in most cases did not interfere with nuclear apparatus staining. Feebly stained cytoplasm and exospore clearly indicated unstained zone of endospore, making it easier to diagnose both mono- and diplokaryotic spores. Staining of extruded polar filament allowed to demonstrate viability and to observe some stages of extrusion process of microsporidian spores.