Staining of DNA-phosphate groups with a mixture of azure A and acridine yellow.

This paper deals with staining of DNA-phosphate groups with a mixture of an equal parts of aqueous solution of azure A and acridine yellow in a 1:1 proportion and also embodies a study of the absorption properties of the stained nuclei. It also embodies results of sequential staining of nuclei stained first with azure A followed by staining with acridine yellow and vice versa, after extraction of RNA with cold phosphoric acid. The results indicate that the absorption peaks of nuclei differ from those of nuclei stained for DNA-aldehyde molecules with azure A-SO2 or acridine yellow-SO2. The in vitro absorption characteristics of an aqueous solution of azure A and those of an aqueous solution of acridine yellow are also presented herein. The conclusion obtained from this study is that all the phosphate groups of DNA do not take part in the staining process when staining is carried out with azure A or acridine yellow alone after after RNA has been extracted. This is because the nuclei stained with these dyes sequentially show the presence of acridine yellow-DNA and azure A-DNA complex.     

Azure B staining of DNA-aldehyde molecules of acid-hydrolysed tissue sections

This communication presents a method for the preparation of azure B-SO2 with trichloracetic acid (TCA) and potassium metabisulphite for in situ demonstration of DNA-aldehyde molecules following acid hydrolysis of tissue sections. The shelf-life of such a dye-reagent is slightly more than that of the control, prepared with N HCl and potassium metabisulphite. The slightly increased shelf-life of the experimental dye-reagent has been considered to be due to a somewhat higher pH as compared with that of the control. The in situ absorption characteristics of nuclei stained for DNA-aldehyde molecules with either an aqueous solution of azure B or with TCA-azure B-SO2 show peak-absorption at 600 nm in both cases. This phenomenon has been interpreted as due to the fact that azure B does not contain any primary amino group in its molecules and, therefore, the mode of binding of DNA-aldehydes with this dye is different from that with dyes that contain primary amino group. The implications of some of the findings have been discussed.     

Preparation of a new red dye from Janus black and its use in the staining of DNA-aldehyde molecules

This communication presents a method for the preparation of a new red dye from an aqueous solution of Janus black by adding NHC1 and sodium thiosulphate to it. This new red dye when used on acid-hydrolysed tissue sections reveals the presence of red nuclei when sections after staining are dried between folds of filter paper, differentiated in n-butanol, cleared in xylene and mounted. Similarly stained sections when treated with SO2 water show partial leaching of the dye from the nuclei. Tissue sections when treated with cold concentrated phosphoric acid for 20 min and then stained with an aqueous solution of Janus black reveal the presence of orange-red nuclei. The new red dye obtained from Janus black does not respond to treatment under UV rays. The in vitro absorption data of the red dye indicate peaks at 210, 270 and 545 nm. The in situ absorption spectra of nuclei stained with the new red dye following Feulgen procedure reveal the peak of maximum absorption at 560 nm and those of nuclei treated with cold concentrated phosphoric acid and then stained with this red dye reveal peak at 530--540 nm. Some relevant points raised out of this investigation have been discussed.     

Staining of phagocytized Cryptococcus neoformans with DAPI

The DAPI (4',6-diamidino-2-phenylindole)-fluorochrome was used to demonstrate phagocytized Cryptococcus neoformans using the mouse peritoneal cavity technique. These yeast cells were chosen because they are large and their capsules exhibit a deep yellow fluorescence which contrasts very well with the blue fluorescent nuclei of the phagocytes (preferentially macrophages). In other words, DAPI stains both, acid mucopolysaccharides and nuclear DNA. The capacity of the phagocyte nuclei to surround or even enclose the yeast cells was the most remarkable result. Generally, the application of DAPI in these phagocytosis experiments provides valuable information rapidly, easily and specifically.     

Staining mycobacteria with periodic acid-carbol-pararosanilin: principle and practice of the method.

Mycobacteria may be acid-fast, non-acid-fast or even chromophobic in staining under different conditions. The pretreatments with oxidants including periodic acid increase effectually the acid-fastness of acid-fast bacilli. This is caused by additional free carboxyl groups resulting from non-acid-fast wax in the cell walls by demethylation with oxidants. Only by prolonged periodic oxidation the aldehyde groups formed as oxidation products of 1-amino-2-hydroxy groups in the cells can be demonstrated with carbol-fuchsin stain. This reaction is most probably attributable to the formation of Schiff's bases between fuchsin and aldehydes. Since pararosanilin is the active molecule in the diphenamine reaction, periodic acid (10%, 24 hours) followed by carbol-pararosanilin stain is a most sensitive and selective method to demonstrate mycobacteria including chromophobic forms.     

激光共聚焦显微镜在新生大鼠耳蜗组织染色中的应用

本文目的是探讨耳蜗组织免疫荧光染色及激光共聚焦显微镜技术的应用.分离新生大鼠耳蜗,培养于胶原支持物上,采用0.25mM的顺铂作用24h,NF-200免疫荧光染色,激光共聚焦显微镜下观察顺铂对耳蜗毛细胞和神经元的毒性作用.结果显示,顺铂对培养的耳蜗毛细胞的损伤没有频率特异性,毛细胞缺失可出现在整个基底膜;谷氨酸盐受体抑制剂-CNQX与顺铂同时作用耳蜗基底膜的神经元损伤程度与单纯顺铂组没有明显差别.结论表明,顺铂能够导致耳蜗毛细胞丢失和形态改变,对耳蜗神经元的损伤可能是顺铂对神经元的直接毒性.     

Effect of the functional groups in ionic liquid molecules on the friction and wear behavior of aluminum alloy in lubricated aluminum-on-steel contact

Four imidazolium-based room temperature ionic liquids containing phosphonyl functional groups, i.e. 1-(3′-O,O-diethylphosphonyl-n-propyl)-3-alkylimidazolium tetrafluoroborates and hexafluorophosphates, were synthesized. The physical properties of the resulting synthetic products were evaluated, and their tribological behaviors as the lubricants for an aluminum-on-steel sliding system were evaluated on an oscillating friction and wear tester, with the emphasis being placed on the effect of the O,O-diethylphosphonyl groups in the ionic liquid molecules on the tribological behaviors. Thus the friction and tests were conducted at a frequency of 25 Hz, a sliding amplitude of 1 mm, and for a duration of 30 min. The worn aluminum surface was analyzed by means of scanning electron microscopy, energy dispersive X-ray analysis, and X-ray photoelectron spectroscopy. It was found that the synthesized ionic liquids had better friction-reducing and anti-wear ability for the aluminum-on-steel system than their nonfunctionalized courterparts (1-ethyl-3-hexylimidazolium tetrafluoroborate, coded as L206, and 1-propyl-3-octylimidazolium hexafluorophosphate, coded as LP308). Especially, they had much better load-carrying capacity than L206 and LP308. The tribological behaviors of the synthetic lubricants were dependent on both the anions and the side-substituted alkyl chains attached to the imidazolium cations. Moreover, physical adsorption and complicated tribochemical reactions were involved during the sliding process of the Al-on-steel system under the lubrication of the synthetic functionalized ionic liquids, which led to the generation of physically adsorbing and chemically reacting films composed of five-member-ring complex compounds, metal fluorides, nitrogen oxide, and FePO₄ on the rubbed Al surface. Those physically adsorbing and chemically reacting films contributed to effectively decrease the friction and wear of the aluminum sliding against steel.     

Multitrack Staining Apparatus with High Resolution and Time-Lapsed Acquisition for Polyacrylamide Gels

Polyacrylamide gel electrophoresis (PAGE) is commonly used for the separation, identification, and purification of mixtures of proteins and nucleic acids. A suitable staining method for the polyacrylamide gels is imperative to determine low concentrations of resolved macromolecules. Several staining methods have been adopted; ethidium bromide, silver, and coomassie staining are the most frequently employed. Silver staining determines macromolecules at nanomolar concentrations but involves many steps and thus is tedious for multiple gels in parallel. In this article, a simple multitrack laboratory-constructed silver staining apparatus is characterized that may enhance the sensitivity of the electrophoresis stains and the homogeneity of detection. In addition, the low cost and simplicity of this system provide wide applicability for gel electrophoresis.     

Staining of biological material for the scanning electron microscope

A histochemical technique has been used in scanning electron microscopy to localize specific areas in biological material. The specific contrast introduced by the staining procedure depends on the interaction of complex variables such as the coating material, the accelerating voltage and the use of a primary or secondary electron signal.     

Staining methods for the detection of laticifers in plant tissue

Simple, easy and inexpensive methods have been evolved to detect laticifers in plants with the help of bright-field and fluorescence microscopy using Oil Red O, dansyl chloride, neutral red, Rhodamine B and Ponceau S stains.     

Nonlinear optical properties of chromophore-containing polyimides with covalently attached dyes

Spectral dependences of quadratic nonlinear coefficients of new chromophore-containing polyimides with covalently attached DR13 chromophore molecules are synthesized and studied by the second-harmonic generation method in the fundamental radiation wavelength from 800 to 1400 nm. The obtained record values of quadratic nonlinear coefficients are 80 to 120 pm/V. Dispersions of refractive indices and extinction coefficients in the spectral range from 400 to 800 nm are determined by spectral ellipsometry, resulting in the improved accuracy of nonlinear coefficients..     

Staining mycobacteria with carbolfuchsin: properties of solutions prepared with different samples of basic fuchsin.

Acid fast staining of mycobacteria in the form of beadings is obtained by means of a carbolfuchsin solution (Ziehl-Neelsen stain) prepared from pararosaniline or from certain kinds of basic fuchsin. After such acid-fast stains, the intensity of the bacilli's colouring was rather poor and unstable, so that some bacilli lost their acid-fast stain. In contrast, an acid-fast staining of mycobacteria in rod form results by using a carbolfuchsin prepared from rosaniline or from other basic fuchsins included new fuchsin. The spectrophotometric and thin-layer chromatographic data indicate that the main component of those basic fuchsins showing beady staining may be pararosaniline, whereas the main ingredient of basic fuchsin with staining the bacteria in rod form may be its higher homologues. Neither chloride nor acetate of the fuchsin could affect the appearance and number of stained bacilli. The commercially available "basic fuchsin" is either the chloride or acetate of pure pararosaniline or consists of variable mixtures of it with higher homologues. Consequently, only a basic fuchsin which has an absorption maximum at lambda greater than or equal to 552 nm could be employed for the acid-fast stain of mycobacteria in a stable manner. Pararosaniline included some basic fuchsins, composed mainly from pararosaniline, should not be selected for the preparation of the carbolfuchsin formula.     

Probing confined fields with single molecules and vice versa

Single dye molecules are used as local probes to map the spatial distribution of the squared electric field components in the focus of a high numerical aperture lens. Simulated field distributions are quantitatively verified by experimentally obtained fluorescence excitation maps. We show that annular illumination can be used to engineer the field distribution in the focus at a dielectric/air interface such that electric field components in all directions acquire comparable magnitudes. The 3D orientation of molecular absorption dipoles can be determined by comparing measured to simulated image patterns. The presence of longitudinal electric field components in a focus is of particular interest in tip-enhanced scanning near-field optical microscopy.     

In situ demonstration of DNA with Schiff-type dyes prepared with meta-phosphoric or tartaric acid

A new method for the preparation of azure A-SO2 and safranine-SO2 For use in Feulgen procedure has been described herein. The method involves the use of m-phosphoric acid or tartaric acid in place of N HCl in the preparation of these eye-reagents which exhibit enhanced pH producing increased staining intensity of the nuclei as compared with those of the controls, prepared with N HCl. Possible explanation for the increased staining intensity as well as the reason for the shorter shelf-life of these eye-reagents have been offered.     

Photoelectron microscopy and photoelectron quantum yields of the fluorescent dyes flourescein and rhodamine

Photoelectric properties of the dyes fluorescein and rhodamine were determined with the aim of assessing the usefulness of these compounds as labels in photoelectron microscopy. The photoelectron quantum yields were measured over the wavelength range 180–230 nm. At 230 nm the quantum yields for fluorescein disodium salt, rhodamine B free base and rhodamine B HCl salt are approximately 10^-5 electrons per incident photon. At 180nm these values rise to approximately 10^-3 electrons per incident photon. All forms of fluorescein do not have the same quantum yield. The neutral form of fluorescein has a quantum yield an order of magnitude lower than the disodium salt. Beam current measurements were performed on labeled and unlabeled proteins to determine the effect of the high light intensity employed in the photoelectron microscope. The initial beam current measurements and the quantum yield curves are consistent and demonstrate that there is significant contrast between labeled and unlabeled proteins. However, after several minutes in the photoelectron microscope, the proteins become more photoemissive and the contrast diminishes. This change in contrast explains several puzzling observations in the literature.     

Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet- and visible-wavelength light

Optical modifications to a confocal scanning laser microscope are described which allow simultaneous fluorescence imaging of living specimens excited by ultraviolet (UV)- and visible-wavelength light. Modifications to a Bio-Rad MRC 600 Lasersharp confocal microscope include the introduction of UV-path-specific lenses and a specially designed UV transmitting eyepiece and tube lens. Upon UV excitation these modifications provide similar resolution and field flatness when compared with visible confocal microscopy. The UV-path-specific optics could be adjusted to correct for varying amounts of longitudinal chromatic aberration in commercially available objectives. Eyepiece and tube lenses were chromatically corrected for UV through visible wavelengths to minimize lateral chromatic error. With these modifications, UV-wavelength light may be used to excite ratioing dyes to quantify intracellular ion concentrations, or as an energy source to release caged compounds in a spatially restricted volume, while simultaneously imaging with dyes excited by visible-wavelength light.