A simple fluorescence staining technique for the differentiation of human tissue transplanted into nude mice.

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during interphase appears as brilliant fluorescent chromocenters. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangements changes according to the stage of differentiation of the cell of the position of a particular nucleus within the cell cycle.     

A Simple Fluorescence Staining Technique for the Differentiation of Human Tissue Transplanted Into Nude Mice

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during inter phase appears as brilliant fluorescent chromo-centers. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangement changes according to the stage of differentiation of the cell or the position of a particular nucleus within the cell cycle.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Retardation of immunofluorescence fading during microscopy

Polyvinyl alcohol (PVA) mounting medium containing paraphenylenediamine (PPD), n-propyl gallate (NPG), or 1,4-diazobicyclo(2,2,2)-octane (DABCO) was compared with PVA alone or buffered glycerol with regard to capacity for preservation of immunofluorescence preparations. The results were based on staining of an artificial substrate with homogeneous antigen distribution followed by microphotometric determination of the initial light emission from bound fluorescein isothiocyanate (FITC)-labeled antibody and the subsequent fluorescence fading during 3-min exposure to blue excitation light. At a concentration of 0.2-2.0 g/liter and 6 g/liter, respectively, PPD and NPG were shown to effectively retard fluorescence fading without notably decreasing the initial emission intensity; two requisites were that the modified PVA used must be rather fresh and that the mounted preparations be examined within a few days. Although addition of DABCO (6 g/liter) afforded a mounting medium that tolerated storage before use better, but both PPD and NPG were more advantageous in practice. The retarding effect of PPD on fading of FITC emission was confirmed by performance testing on human tissue sections. Remounting in PVA alone is recommended for prolonged storage of sections that have been mounted in PVA modified with one of the above-mentioned compounds.     

Introduction of antibody into viable cells using electroporation

Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.     

Rapid Preparation of Thick Sections of Fleshy Plant Tissues

Methods are described for permanent micro-slide preparations of soft, large-celled plant tissues such as ripe fruit. Thick sections (200-800 [t) cut on a sliding microtome are aspirated in an aqueous killing agent; after fixing and washing, the sections are dehydrated and cleared in an alcohol-xylene series. Infiltration with 20, 30, and 40% solutions of mountant prior to mounting the sections is necessary to avoid too abrupt changes in the cleared tissues. Several staining methods have been successfully used for different purposes. The final preparations showed nearly perfect preservation of intact cells and intercellular spaces in their 3-dimension-al structure.     

Synthesis and spectral properties of DNA capped CdS nanoparticles in aqueous and non-aqueous media

Synthesis of novel cadmium sulphide nanoparticles has been carried out in aqueous and non-aqueous media. DNA has been added during the synthesis of the nanoparticles, which results into cadmium-rich nanoparticles forming a stable complex with DNA. These particles exhibit strong fluorescence, spectral nature of which depends upon the medium in which the particles are synthesized. When interacted with proteins, fluorescence peak intensity of CdS nanoparticles increases considerably. It is possible that such CdS nanoparticles would be useful as a protein sensor.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Methods for taxonomic and distributional studies of terrestrial flatworms (Tricladida: Terricola)

Traditionally used methods for collecting and preserving terrestrial flatworms have proved deficient in various respects. Comparison of methods for preparing these animals for taxonomic study has shown that preservation of morphological characters can best be achieved using formaldehyde-calcium-cobalt fixative and subsequent storage in an aqueous solution of propylene phenoxetol plus propylene glycol and that the best-lasting histological preparations make use of light-fast, oxidation-resistant dyes and synthetic mounting media with an anti-oxidant.     

Microscopical and spectroscopic studies on the fluorescence of a daunomycin-aluminum complex.

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566 nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560 nm under optimal excitation at 470 nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580 nm (optimal excitation at 530 nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.     

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.     

A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.     

An Improved Mounting Method for Observation of Thick Specimens Using Confocal Microscopy

When applying aqueous media to mount thick specimens, air bubbles occur frequently between coverslips and slide glasses. This results in an increase of background signal by confocal microscopy. Using Spurr's resin as a mounting medium, we could observe thick specimens with oil immersion objective lens without the use of coverslips, then avoid air bubbles near the specimen. Fading of the fluorescence appeared to be less in specimens mounted by the resin than in specimens mounted by aqueous media. Further, the new method increased depth of field so that more planes of specimens could be analyzed.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

An Ultraviolet-Schiff Reaction for Unsaturated Lipids

Tissues are fixed 12-18 hr in cold, neutral, 10% aqueous formalin, sectioned on a freezing microtome and the sections placed in a shallow, open dish of water under an ultraviolet light source. Sections are then treated with Schiff's reagent for 15 min, rinsed in 3 changes of sulfurous acid (3 min in each) and in distilled water and mounted in an aqueous mounting medium. The time of irradiation necessary to produce a maximum reaction is determined empirically. The magenta color indicating the site of unsaturated lipids fades and runs in many preparations in a few days. Evidence is presented that this reaction is most likely the histochemical equivalent of an iodine number determination.     

Fluorescence in Paraffin Sections of Dye-Labelled Protein Administered in vivo: Effect of HgCl2 Fixation

Lung and liver slices, 2-3 mm thick, from guinea pigs injected intravenously with fluorescent dye-protein conjugate are fixed for 15-30 min in saturated aqueous HgCl₂, dehydrated in ethanol, cleared in xylene and embedded in paraffin at 60 C. Mercurial deposits are removed with I₂KI from 5 μ sections taken to water, and the iodine then removed with 5% Na₂S₂O₃. Sections are mounted from xylene into permanent nonfluorescent mounting medium. This procedure gives optimal fluorescence which is not decreased by the technic of removing mercurial precipitates. Longer fixation, fixation in phosphate-buffered formalin, or in an HgCl₂-formalin mixture gives inferior results.     

Experiences with improved preservation solutions for historic and modern anatomic preparations

Basing on experiences made at the Anatomical Museum Basel often arising difficulties of conservation of ancient anatomical preparations are described. Criteria are named which allow the recognition of specific problems of various preparations, and the adequate solutions for fixation and conservation are given. These solutions, the compositions of which are described in detail, are suited for historical as well as for modern wet preparations. Thus, it is possible to save and stabilize also delicate preparations of former times, even those with faint hues can be renovated which afterwards show an increase of contrast and an improved preservation of colours. Furthermore, techniques are indicated for mounting and preservation of preparations in special containers made of acrylic glass. By means of these methods, the preservation of preparations as well as the cleaning and change of solutions are markedly improved.     

Fluorescent C bands of human chromosomes with 33 258 Hoechst stain

Summary Air-dried preparations of human metaphase chromosomes normally exhibit a Q band fluorescence pattern with 33 258 Hoechst stain while the C band regions of 1, 9, 16, and most acrocentric short arms appear dull. If these stained and mounted slides are stored at room temperature in the dark for several days, a spontaneous change from C-negative to C-positive bands sometimes occurs. We postulate that the pH of the buffered saline mounting medium during storage of the slides causes the C band shift since the results can be duplicated experimentally by lowering the pH of the mounting buffer from 5.5 to 4.0.     

An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy

We present a new procedure for effectively detaching particle-associated bacteria by 10% (v/v) methanol and sonication which is particularly suitable for samples with a high particle load and sediments. We also optimized the sample preparation by applying the highly dsDNA-specific fluorescent stain SybrGreen I together with an optically brilliant mounting medium (polyvinylalcohol 4-88, 'moviol') in one step. The new protocol allows a much faster, easy and less toxic handling of samples as compared to other methods. Cells are stained directly on a black Nuclepore filter and show an intensive fluorescence signal with low background. The detachment procedure was optimized with respect to the temperature of the 10% methanol solution (35 degrees C), ultrasonication and centrifugation. The application of the new method in comparison with detachment procedures with pyrophosphate and Tween-80 with various types of marine samples including sediments always yielded higher numbers and/or higher fractions of particle-associated cells. Staining and mounting the samples with the moviol-SybrGreen I solution allowed an accurate and highly reproduceable enumeration of bacteria also in samples with high concentrations of SPM. Fixation of bacteria by glutardialdehyde resulted in a brighter fluorescence as compared to fixation by formalin. Because of the high specificity to dsDNA and bright fluorescence of SybrGreen I, the fast and easy handling and the possibility to store stained samples for at least several months at -20 degrees C without any loss in fluorescence intensity, the newly developed method is also an attractive alternative to DAPI staining of aquatic bacteria.