bluuebunny/crossref_metadata_2025_split huggingface dataset:
Image processing of immunogold-labeled titin filaments in skeletal muscle
Authors: Ramirez-Mitchell R. , Wright John , Wang Kuan 
Published: June 2020

In our electron microscopic studies of the elastic behavior of titin filaments in skeletal muscle sarcomeres, immunogold techniques have been used to detect the elastic stretch-response of titin epitopes in the I-band region. The use of gold conjugates was essential, since in highly stretched muscles, distortion of the sarcomere led to the loss of lateral registration of epitopes. In such cases, antibody densities are smeared and difficult to detect. We observed, however, that gold beads sufficiently small ( < 5 nm ) to penetrate the I-band lattice were often obscured by electron dense stains. To overcome this technical problem, we developed a procedure using image processing techniques to differentiate and identify gold beads.In the following example, titin filaments were rendered visible by stretching rabbit semitendinosus muscle using a split-fiber technique to a length where myosin and actin filaments no longer overlap. The titin filaments in the gap region were labeled by monoclonal anti-titin (RT13) followed by protein A-gold conjugates.
The identifying of intermediate filament-lamina-nuclear matrix system using immunogold labeling
Authors: Zhonghe Zhai , Renjie Jiao , Donglan Wu , Bo Zhang , Shutao Cai 
Published: June 2020

The immuno — gold labelling technique was used in our work in combined with the use of monoclonal anti — keratin, vimentin, lamin A, and 298KD nuclear matrix protein antibodies to label the selective — extracted whole mount HeLa cells and BHK-21 cells.The cells were grown on Formvar \carbon— coated nickle grids in Eagle’s MEM medium and extraction procedures was essentially according to Fey’s method with some modifications. Treatment first with 0.5 % Triton X — 100 to remove membrane system and some soluble proteins, then with 1 % Tween 40 and 0.5 % sodium deoxycholate to strip microtubules and microfilaments. DNase I and ammonium sulfate was added to remove chromatin and histone. For electron microscopy, above samples were prefixed in 0.25% glutaraldehyde, then incubated with monoclonal antibodies at 37 °C for 1hr, washed with PBS and then incubated with diluted protein A — gold solution at 37 °C for 1hr. The samples were conventionally fixed, dehydrated, dried through CO2 critical point.
Immunocytochemical methodology for the localization of neuronal antigens at the ultrastructural level
Authors: Pecci Saavedra Jorge , Connaughton Mark , José López Juan , Brusco Alicia 
Published: June 2020

The use of antibodies as labels for the localization of specific molecules in the nervous systan has been extensively applied in recent years. Both monoand polyclonal antibodies or antisera have been employed. The knowledge of the organization of neuronal connectivities, gliovascular relationships, glioneuronal relationships and other features of nerve tissue has greatly increased.A number of areas of the nervous systan have been analyzed in our laboratory, including the nuclei of the raphe system, the reticular formation, interpeduncular nucleus, substantia nigra, caudate nucleus, putamen, pallidum, spinal cord, pineal gland and others.From a technical point of view, a number of variables needed to be taken into account in order to obtain reliable and reproducible results. The design of the optimal conditions of tissue fixation, embedding, sectioning, dilution of antibodies, and adaptation of Sternberger PAP technique were sane of the parameters taken into account to optimize the results. It is critical that each step of the technique be defined for each particular case.
High time resolution enzyme cytochemistry of rod outer segment
Authors: Saito Takuma , Takizawa Toshihiro 
Published: June 2020

Cells and tissues live on a number of dynamic metabolic pathways, which are made up of sequential enzymatic cascades.Recent biochemical and physiological studies of vision research showed the importance of cGMP metabolism in the rod outer segment of visual cell, indicat ing that the photon activated rhodopsin exerts activation effect on the GTP binding protein, transducin, and this act ivated transducin further activates phosphodiesterase (PDEase) to result in a rapid drop in cGMP concentration in the cytoplasm of rod outer segment. This rapid drop of cGMP concentration exerts to close the ion channel on the plasma membrane and to stop of inward current brings hyperpolarization and evokes an action potential.These sequential change of enzyme activities, known as cGMP cascade, proceeds quite rapidly within msec order. Such a rapid change of enzyme activities, such as PDEase in rod outer segment, was not a matter of conventional histochemical invest igations.
An immunoelectron microscopic study of T-cell lymphomas, with special reference to Japanese adult T-cell lymphomas
Authors: Shamoto Mikihiro 
Published: June 2020

A detailed ultrastructural morphology of antigen-antibody reactions using monoclonal antibodies is very valuable, not only for accurate diagnosis, but also for the understanding of the character of malignant lymphomas. This is a report of ultrastructural immuno-histochemical analysis of T-cell lymphomas, with special reference to Japanese T-cell lymphomas (ATL).Biopsy specimens of lymph nodes from 30 patients with T-cell lymphomas, (including 20 patients with ATL) were used. Frozen sections of 10 u thickness were cut from specimens fixed in periodate-lysine-paraformaldehyde. The available antibodies were, OKT 3/Leu 4, OKT 4/Leu 3a, OKT 8, Tac, and FTF 148. The sections were placed on slides, and overlayed with appropriate diluted monoclonal antibodies and incubated for 2 hours. Then they were incubated with biotinylated horse anti-IgG for 2 hours, and in both an avidin-biotinylated peroxidase complex and a peroxidase mouse anti-peroxidase complex for 2 hours. They were fixed in 2% glutaraldehyde, then incubated with DAB hydrogen peroxide, and postfixed in 2% OsO4. The specimens were embedded in inverted gelatin capsules and cut with a diamond knife.
A combined pseudoreplica-immunocytochemical technique for research and diagnostic virology
Authors: Fritz Blayne , Naides Stanley J. , Moore Kenneth 
Published: June 2020

The pseudoreplica method of staining viral particles for visualization by transmission electron microscopy is a very popular technique. The ability to concentrate clinical specimens while semi-embedding viral particles makes it especially well suited for morphologic and diagnostic virology. Immunolabelling viral particles with colloidal gold is a technique frequently employed by both research and diagnostic virologists. We have characterized a procedure which provides the advantage of both by modifying and combining pseudoreplica staining and immunogold labelling.Modification of specimen retrieval and delay of staining allows us to utilize pseudoreplica processed specimens within our standard immunogold labelling protocol. In brief, we absorbed samples onto 2% agarose, added.25% Formvar and wicked dry. We then floated the Formvar-virus film onto double distilled water, added grids and retrieved with parafilm. The Formvar-virus specimens were then treated as thin tissue sections within our standard two stage immunolabelling protocol. Following completion of immunogold labelling; each grid was negatively stained with phosphotungstic acid or uranyl acetate contrast stains.
Immunocytochemical localization of xanthine oxidase in rat hepatocytes processed by quick freezing followed by freeze-substitution fixation
Authors: Ichikawa Misao , Ichikawa Atsushi , Nishino Takeshi , Nishino Tomako 
Published: June 2020

Quick freezing of tissues, with freeze-substitution fixation, not only provides superior ultrastructural detail in cells and tissues but also retains soluble substances within their normal intracellular compartments. Combining this procedure with postembedding protein A-gold immunocytochemical technique permits precise localization of antigenic sites at the electron microscopic level.Biochemical and cytochemical studies have demonstrated variously that xanthine oxidase, one of the flavoprotein enzymes involved in urate metabolism, is localized in cytozol, peroxisomal membrane, and/or peroxisomal crystalline cores of rat hepatocytes. This study is designed to determine the localization of xanthine oxidase in rat hepatocyte by high resolution immuno-electron microscopy.Adult Wistar rats, 150-230 g, maintained on standard rat food pellets and water ad libitum were fasted for 12 h before sacrifice. To examine the presence of xanthine oxidase in peroxisomes in rat hepatocyte, some animals were treated with intramuscular injections of 250 mg (per day) of clofibrate, a hypolipidemic drug, for 10 days to generate peroxisome proliferation. Fresh tissue blocks were quickly frozen by the metal contact method, using liquid helium, and then freeze-substituted with either 2% OsO4 in acetone or 0.3% glutaraldehyde-acetone solution.
Cytochemical staining of connective tissue extracellular matrix with ruthenium hexammine trichloride (RHT)
Authors: Bray Douglas F. 
Published: June 2020

The extracellular matrix (ECM) of fibrous connective tissue is a composite of protein- and carbohydrate-containing structures. Preservation of the protein components is achieved through fixation with glutaraldehyde and osmium tetroxide. Carbohydrates however are inadequately preserved and often lost using these fixatives. Cationic dyes such as ruthenium red, alcian blue, and several others have been used to stabilize and retain carbohydrates, particularly the proteoglycans (PGs), but usually with less than optimal results. The present study documents the improved preservation and staining of PGs and other carbohydratecontaining components of skin ECM using ruthenium hexammine trichloride (RHT).Human neonatal foreskins were cut into 1 mm 3 blocks, fixed for 3 h in cacodylate buffered (0.1M, pH 7.2) 2.5% glutaraldehyde containing 1 mg/ml RHT (Polysciences, U.S.A.), postfixed 1 h in buffered 1% OsO4 also containing 1 mg/ml RHT, dehydrated in ethanol and embedded in Spurrs. In some cases the tissue was digested with chondroitinase ABC (Miles, U.S.A.) prior to fixation. Sections from tissue fixed without RHT were poststained in uranyl acetate and lead citrate. Figs. 2 - 5 are from 0.5 μm sections of tissue immunolabelled with monoclonal antibodies to type VI collagen.
Use of a novel colloidal gold probe to assess the distribution of Na,K-ATPase following covalent association in a variety of cell types
Authors: Bork Tracie , Mrsny Randall J. 
Published: June 2020

Na,K-ATPase is an integral membrane protein complex capable of actively transporting Na+ and K+ ions. Function of this enzyme is essential for maintenance of cellular ion homeostasis and plasma membrane potential. Ouabain, a cardiac glycoside, has been demonstrated to selectively inhibits the transport ionic transport activity of Na,K-ATPase. Several soluble and membrane associated factors have been shown to modify the function of this transport enzyme by a variety of mechanisms. Recently, evidence has been obtained which suggests a movement of Na,K-ATPase from intracellular stores to the plasma membrane and back again, possibly in response to cellular events and in a fairly rapid time frame.A number of methods including immunolabeling, tritiated ouabain autoradiography, and K+-dependent nitrophenylphosphate cytochemistry have been successfully employed to examine the distribution of Na,K-ATPase. We have used yet another approach, termed affinity-gold labeling, to localize the Na, K-ATPase of a variety of cell types. This approach, which employs a receptor-specific ligand and the potential for covalent association of the colloidal gold-containing probe to the receptor following association, has the added advantages of rapid identification of the labeled structure(s) and the potential for nearest-neighbor assessment for the receptor being labeled.
High-voltage cytochemistry for three-dimensional observation of nuclei and calculation of total numbers of nuclear pores in retinal Mueller glia
Authors: Ishigooka H. , Ueno S. , Hjelmeland L.M. , Landers M.B. , Ogawa K. 
Published: June 2020

Introduction: We have demonstrated that Glucose-6-phosphatase (G6Pase) activity is localized to the endoplasmic reticulum and nuclear envelope of Mueller glia in the normal and pathological guinea pig retina. Using a combination of this cytochemical technique and high voltage electron microscopy, the distribution of nuclear pores could be clearly observed on the nuclear envelope of Mueller glia because of their anatomical lack of reaction products. This technique was developed to study the three-dimensional structure of nuclei and to calculate total numbers of nuclear pores utilizing a computer graphic analysis system in the normal and pathological retina.Materials and methods: Normal and photocoagulated retina of pigmented adult guinea pigs were perfused with a cold mixture of 0.25% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, and the enucleated globes were hemisected and immersed in the same fixative for 30 min. After sectioning and incubation in the reaction medium for the detection of G6Pase activity by the method of Wachstein-Meisel, the sections were postfixed, dehydrated and embedded in Spurr’s epoxy resin. Serial thick sections (1.0um) were prepared for the observation by a Hitachi high voltage electron microscope (H 1250-M) with an accelerating voltage of 1000 Kv. and pictures were analyzed and three-dimensionally reconstructed by TRI (RATOC Co., Ltd.).
Cell surface changes during aging of human bladder epithelium examined by lectin probes and SEM
Authors: Jacob J. , Ismail M.F.M. 
Published: June 2020

Ultrastructural changes have been shown to occur in the urinary bladder epithelium (urothelium) during the life span of humans. With increasing age, the luminal surface becomes more flexible and develops simple microvilli-like processes. Furthermore, the specialised asymmetric structure of the luminal plasma membrane is relatively more prominent in the young than in the elderly. The nature of the changes at the luminal surface is now explored by lectin-mediated adsorption visualised by scanning electron microscopy (SEM).Samples of young adult (21-31 y old) and elderly (58-82 y old) urothelia were fixed in buffered 2% glutaraldehyde for 10 m and washed with phosphate buffered saline (PBS) containing Ca++ and Mg++ at room temperature. They were incubated overnight at 4°C in 0.1 M ammonium chloride in PBS to block any remaining aldehyde groups. The samples were then allowed to stand in PBS at 37°C for 2 h before incubation at 37°C for 30 m with lectins. The lectins used were concanavalin A (Con A), wheat germ agglutinin (WGA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) at a concentration of 500 mg/ml in PBS at pH 7.A.
Analysis of Gene Expression Directed by a Thymic Locus Control in Transgenic Mice: Mechanisms and Application to Vectors for Gene Therapy
Authors: Aronow BJ , Ley CA , Ess KC , Witte DP 
Published: June 2020

The formation of peripheral T cells from thymocyte progenitors is an intricate developmental process that requires the organized and coordinate expression of multiple genes. Adenosine deaminase (ADA) is an example of a gene that is subject to strong developmental regulation in T-cell precursors and is essential for the subsequent formation of T-cells in humans. We have sought to understand the mechanisms of ADA gene regulation from a basic point of view as well as to employ this to potential vectors for gene therapy.Using transgenic mice have shown that the first intron of the ADA gene contains a powerful locus control region that directs high level gene expression within cortical thymocytes. Based on extensive mutational analysis of the regulatory region and analyses of gene expression that include quantitative gene expression, in situ hybridization, and biochemical characterizations of chromatin structure, we have demonstrated that the intronic locus control region (LCR) consists of a hierarchically structured 2300 base pairs of DNA sequence (Figure 1). The LCR is composed of a series of regulatory elements that include a centrally positioned 300 base pair classical enhancer domain within which there is a critical 30 base pair enhancer core. Within this core, there is a single binding site for the transcription factor c-Myb that is required for activity of the enhancer core, the enhancer, and the intact LCR. Beyond the 300 bp enhancer core on either side the LCR contains novel and puzzling 1 kb non-enhancer sequences that we have termed facilitators. These sequences enable gene copy proportional expression by facilitating the ability of the enhancer to function in chromatin. The effects of the facilitators are evidenced by their ability to allow for insertion-site-independent and gene-copy-proportional expression and they prevent variegated expression among similarly differentiated cell types (Figure 2). Thus, total gene expression does not indicate proper cell type specific expression. The facilitators also allow for the formation of a strong tissue and cell type specific DNAse I hypersensitive site at the enhancer. This suggests that the formation of a discrete organized chromatin structure as a function of developmental differentiation requires extensive DNA sequences, only some of which are of the enhancer type. The capabilities of the facilitators to activate a chromatin domain may also suggest their potential usefulness in vectors for gene therapy of both ADA deficiency and possibly other human genetic diseases. However, the distance and non-enhancer nature of the facilitators suggest that they may act differently than conventional regulatory elements. In support of this, the facilitators obey a strict position and orientation rules with respect to the enhancer.
Stomatal morphology and distribution of Pinus koraiensis
Authors: Ning Zhu Hua , Abdollahi Kamran Ken 
Published: June 2020

Stomata are the important channels for the necessary exchange of gases between the plants and the surrounding earth. Stomata open and close in response to environmental and physiological signals, thus helping the plant maintain a balance between its water losses and its oxygen and carbon dioxide requirements. This paper reports the study on stomatal morphology and distribution of Pinus koraiensis, a five needle pine tree with a natural distribution in Northeastern China.Needle samples were collected from one-year-old seedlings. They were cut into 1mm lengths and fixed overnight at 4°C in 4% glutaraldehyde buffered with 0.07M cacodylate at pH 7.2-7.4. After being rinsed with the same buffer, they were post-fixed for 1 hour in 1 % osmium tetroxide in the same buffer and dehydrated in a graded ethanol series. Afterward, tissues were dried in a DCP-1 critical point dryer and coated in a Hummer II sputter coater. Stomatal morphology and distribution were examined under a Hitachi S-405A scanning electron microscope.
Energy Filtered Electron Backscattering Images of 10-nm NbC and AIN Precipitates in Steels Computed by Monte Carlo Simulations
Authors: Gauvin Raynald , Drouin Dominique , Hovington Pierre 
Published: June 2020

In modern materials science, it is important to improve the resolution of the Scanning Electron Microscope (SEM) because small phases play a crutial role in the properties of materials. The Transmission Electron Microscope (TEM) is the tool of choice for imaging small phases embedded in a given matrix. However, this technique is expensive and also is slow owing to specimen preparation. In this context, it is important to improve spatial resolution of the SEM.In electron backscattering images, it is well know that the backscattered electrons have an energetic distribution when they escape the specimen.The electrons having loss less energy are those which have travelled less in the specimen and thus escape closer to the electron beam. So, in filtering the energy of the backscattering electron and keeping those which have loss only a small amount of energy to create the image, a significant improvement of the resolution of such images is expected. New detectors are now under development to take advantage of this technique of imaging.
Characterization of schottky depletion zone using EBIC imaging
Authors: Drouin D. , Gauvin R. , Beauvais J. , Hovington P. , Joy D. C. 
Published: June 2020

Scanning Electron Microscopy (SEM) has been widely used to characterize semiconductor materials. Approximately one half of all SEMs are used by the electronic industry. The SEM can be used as a local current source that can be scanned over semiconductor device surfaces. This imaging mode is called Electron Beam Induced Current (EBIC).A depletion zone formed by a Schottky diode is visualized using this technique. This Shottky diode is fabricated by placing a metal contact on a semiconductor surfaces. The work function of the metal and the semiconductor produce an equilibrium contact potential. This potential barrier occurs across a region free of charge carriers and thus an electric field is present. This region is called a depletion zone.
A new method for characterization of domain morphology of polymer blends using RuO4 staining and LVSEM
Authors: Brown G. M. , Butler J. H. 
Published: June 2020

Ruthenium tetroxide (RuO4) staining for TEM is a well proven technique for the characterization of crystalline polyolefins. Blend morphology has also been studied using RuO4 staining and SEM and low voltage SEM. A new method has been developed which uses RuO4 staining and LVSEM in the characterization of polyolefin blend morphology, specifically blends of polypropylene modified by the addition of elastomers or plastics. This method is often preferred over TEM for the characterization of blends and is applicable to many problems encountered in commercial and industrial laboratories including the analysis of domain morphology in molded parts, extruded films and fibers, failure analysis, and the analysis of layer morphology in certain coextruded films.Three sample preparation steps are required prior to the LVSEM analysis. (1) The sample face is cryogenically sectioned on a glass knife in the cryomicrotome. (2) This face is stained in RuO4 vapors for 2.5 hours. The staining solution is prepared in situ by a modification of the method of Montezinos.
XRMF applications of a monolithic, polycapillary, focusing x-ray optic
Authors: Carpenter D. A. , Gao Ning , Havrilla G. J. 
Published: June 2020

A monolithic, polycapillary, x-ray optic was adapted to a laboratory-based x-ray microprobe to evaluate the potential of the optic for x-ray micro fluorescence analysis. The polycapillary was capable of collecting x-rays over a 6 degree angle from a point source and focusing them to a spot approximately 40 µm diameter. The high intensities expected from this capillary should be useful for determining and mapping minor to trace elements in materials. Fig. 1 shows a sketch of the capillary with important dimensions.The microprobe had previously been used with straight and with tapered monocapillaries. Alignment of the monocapillaries with the focal spot was accomplished by electromagnetically scanning the focal spot over the beveled anode. With the polycapillary it was also necessary to manually adjust the distance between the focal spot and the polycapillary.The focal distance and focal spot diameter of the polycapillary were determined from a series of edge scans.