Epoxy embedding of thin-layer material in commercially available vinyl cups for light and electron microscopy.
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Epoxy Resins↗
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Exploitation of phylogenetic distance in cell surface immune labeling: studies with beta 2-microglobulin.
Beta 2-microglobulin (beta 2M) is part of the HLA molecule, and is found on the cell surface of human nucleated cells. In certain skin tumors, malignant change has been associated with a loss of this surface beta 2M, indicating a possible diagnostic value for this marker. At present beta 2M is best identified in paraffin-embedded tissue by means of a triple-layer peroxidase-antiperoxidase technique, using mammalian polyclonal antisera. Recently a polyclonal antiserum against human beta 2M has been produced in chickens. Because of the phylogenetic differences between the species, the resulting antiserum is likely to recognize more epitopes on the beta 2M and show greater sensitivity than antisera raised in mammalian species. To confirm this hypothesis, the avian antiserum was compared to both mammalian polyclonal (rabbit) and monoclonal (mouse) antibody in vitro. beta 2M fixed to plastic surfaces combined with more avian than mammalian antibody. Furthermore, insolubilized chicken antibody could bind more secondary antibody-horseradish peroxidase conjugate than could insolubilized mammalian antibody, thus showing even greater enhancement with this system. Immunohistochemical analysis of these systems confirmed that the chicken strategy has greater sensitivity, and can be used in an indirect system with consequent reduction in nonspecific background activity. It is the most suitable technique for the investigation of the distribution of beta 2M in paraffin-embedded tissue.
Animals↗
Structural plasticity of an immunochemically identified set of honeybee olfactory interneurones.
Using a monoclonal antibody (FB 45) raised by Dr. A. Hofbauer (Würzburg) against Drosophila brain we investigated the development and plasticity of immunoreactive cells belonging to the median and lateral antennoglomerular tracts (AGTS) in the honeybee brain. In early stages of pupal development presumed AGT immunoreactivity was detected in the diffuse central neuropil of the antennal lobe as well as in the glomeruli, which differentiate at 40% pupal development. The lateral protocerebral lobe--one target area of the AGTs--is labelled throughout pupal life whereas labelling in the calyces is first restricted to the basal ring region. Although the lips of the calyces develop in middle-aged pupae, they do not show immunoreactivity until the last day of metamorphosis. Unilateral ablation performed on pupae of different stages resulted in size reduction of the antennal lobe and fusion of glomeruli. The number of labelled somata and glomeruli in the antennal lobe were reduced on the treated side. These effects were more prominent when ablation was performed in young pupae. No differences in staining intensity at the light microscopic level were found in the calyces. Therefore a pre-embedding immunohistological approach was developed to detect AGT profiles in the mushroom body at the electron microscopic level.
Animals↗
The fit of fixed partial dentures joined by infrared soldering.
This study determined the accuracy of fit of three-unit fixed partial dentures joined by an infrared soldering technique compared with one-piece fixed partial denture castings and individually cast crowns. Wax patterns of prepared Ivorine teeth, maxillary left central incisor and maxillary left canine, were injection molded; a plastic rod was used as a pontic. One group of patterns was cast as one-piece dentures; the other group was sectioned in the connector area, cast individually and then joined by infrared soldering. Castings were seated on their respective dies, embedded in epoxy resin, and sectioned. Gap distances between the casting and the die were measured at specified marginal sites with a profile projector. Results showed that the fit of infrared-soldered fixed partial dentures was significantly better than that of one-piece castings and was comparable with the fit of single crowns. The gap openings measured in all castings were within the range of clinical acceptability.
Crowns↗
A new technique for separation of coverglass substrate from epoxy-embedded specimens for electron microscopy.
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Animals↗
Cytoplasmic inclusion bodies are detected by synthetic antibody in ciliated bronchial epithelium during acute Kawasaki disease.
BACKGROUND: In developed nations, Kawasaki disease (KD) is the most common cause of acquired heart disease in children. An infectious etiology is likely but has not yet been identified. We have previously reported that oligoclonal immunoglobulin A plasma cells infiltrate acute KD tissues and that synthetic KD antibodies detect a distinctive spheroidal antigen in acute KD ciliated bronchial epithelium. METHODS: To further characterize the antigen in acute KD bronchi, we examined paraffin-embedded ciliated bronchial epithelium using light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: The spheroids observed by immunohistochemistry (IHC) are visualized as inclusion bodies with hematoxylin-eosin and nucleic acid stains and in methylene blue/azure II/basic fuchsin trichrome-stained plastic sections, suggesting the presence of both protein and nucleic acid. The structures visualized by LM correspond to homogeneous electron-dense perinuclear inclusion bodies (up to 1.4 microns in diameter) in ciliated bronchial epithelium from 4 patients with acute KD examined by TEM. Inclusion bodies were not present in control bronchial epithelium or in nonciliated cells. CONCLUSIONS: The antigen detected in acute KD ciliated bronchial epithelium by IHC with synthetic KD antibodies resides in cytoplasmic inclusion bodies that are consistent with aggregates of viral proteins and associated nucleic acid and may derive from the etiologic agent of KD.
Antibodies, Monoclonal↗
Cortical dynamics.
Our ability to interpret visual scenes involves assembling the components of objects into a unified percept and segregating them from background, storing information about earlier visual experiences and testing our interpretations of the visual world against incoming sensory input. The influences of context, experience and expectation are reflected in the response properties of cells at early stages in the visual pathway. The responses of cells in primary visual cortex depend not only on the attributes of features lying within the classical receptive field but also on the global characteristics of the contours and surfaces within which these features are embedded. The substrate for lateral interactions includes a plexus of long-range horizontal connections within each cortical area that links columns of similar orientation specificity and cells with widely separated receptive fields. The horizontal connections also play a role in the plasticity of receptive field structure and of cortical functional architecture, induced over long time scales by retinal lesions and over short time scales by patterned visual stimulation. The long-term changes are associated with synaptogenesis; and the short-term changes with an increase in synaptic effectiveness. The dynamics of cortical function persist throughout adulthood and are general to all cortical areas, including primary sensory cortices.
Animals↗
Synchrony detection and amplification by silicon neurons with STDP synapses.
Spike-timing dependent synaptic plasticity (STDP) is a form of plasticity driven by precise spike-timing differences between presynaptic and postsynaptic spikes. Thus, the learning rules underlying STDP are suitable for learning neuronal temporal phenomena such as spike-timing synchrony. It is well known that weight-independent STDP creates unstable learning processes resulting in balanced bimodal weight distributions. In this paper, we present a neuromorphic analog very large scale integration (VLSI) circuit that contains a feedforward network of silicon neurons with STDP synapses. The learning rule implemented can be tuned to have a moderate level of weight dependence. This helps stabilise the learning process and still generates binary weight distributions. From on-chip learning experiments we show that the chip can detect and amplify hierarchical spike-timing synchrony structures embedded in noisy spike trains. The weight distributions of the network emerging from learning are bimodal.
Action Potentials↗
Alpha isoform of calcium-calmodulin dependent protein kinase II (CAM II kinase-alpha) restricted to excitatory synapses in the CA1 region of rat hippocampus.
Pre-embedding immunoperoxidase staining for CAM II kinase-alpha and post-embedding immunogold staining for glutamate and GABA, were used to reveal the subcellular distribution of CAM II kinase-alpha at transmitter-characterized synapses in the CA1 region of rat hippocampus. Immunoelectron microscopy showed that the majority of CAM II kinase-alpha-immunostained neuronal profiles were dendritic spines presumably derived from pyramidal cells. CAM II kinase-alpha immunoreactivity was mainly localized in postsynatic densities associated with glutamatergic axon terminals. No CAM II kinase-alpha immunoreactivity was detected in GABA-immunoreactive profiles or at GABAergic synapses. This study provides morphological evidence that CAM II kinase-alpha is involved only in excitatory neuronal transmission in the CA1 region. The enzyme is unlikely to be involved in plasticity at GABA synapses.
Animals↗
Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit.
Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.
Adenosine Triphosphate↗
The microwave oven in temporal bone research.
The microwave oven has recently been introduced in morphological research. It can be used for virtually all procedures in histological processing; fixation, dehydration, embedding, staining, immunotechniques, and the techniques used for electron microscopy. Here we report that the microwave oven histoprocedures also have several advantages in temporal bone research on animals. The microwave oven has been tested for (i) fixation, (ii) decalcification, (ii) staining of semithin plastic sections. The microwave oven procedures are time-saving and results in an excellent preservation of the tissues. The microwave histoprocessing should also be applicable to temporal bone research in human.
Animals↗
Subdermal implants of poly(L-lactic acid) with plasticizer: an ultrastructural study in rats.
Poly(L-lactic acid) (PLLA) membranes containing 7% triethylcitrate plasticizer were implanted in the subcutaneous tissue of rats, and the cellular reaction was evaluated over a period of 2-180 days. The samples were processed for conventional transmission electron microscopy. Polymorphonuclear-type cells and a fibrin network were seen within membrane pores 2 days after implantation. In subsequent samples, there was cellular infiltration, which consisted mainly of fibroblasts, macrophages and multinuclear giant cells embedded in an abundant extracellular matrix containing a network of collagen fibers and blood vessels. At 90 and 180 days after implantation, a high density of voluminous phagocytic cells with a large number of endocytic polymer fragments within their cytoplasm was seen. These results show that PLLA membranes can support connective tissue proliferation and remodeling, which are important properties for successful bio-protheses.
Animals↗
Preparation of cultured mammalian cells for transmission and scanning electron microscopy using Aclar film.
Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.
Animals↗
A fine structural study of the cells that proliferate in the partially denervated dentate gyrus of the rat.
Tritiated thymidine autoradiography has established that after interrupting the commissural afferents to the dentate gyrus a number of non-neuronal cells proliferate in the molecular layer. In the present study the fine structure of the proliferating cells was analyzed by reembedding the 2-microns thick plastic sections of the dentate gyrus which had been previously coated with a nuclear emulsion and processed for light microscopic autoradiography. The location of the labeled cells was plotted with a camera lucida and a few ultrathin sections were taken from the re-embedded sections. In these the labeled cells were re-identified and photographed in an electron microscope. Most of the identified proliferating cells exhibited the following morphological features: The nuclei were irregularly oval, sometimes with deep indentations and contained dense clumps of chromatin; their diameters ranged between 4.5 and 6.5 microns. The cytoplasm was generally disposed to one side of the nucleus and often extended into a few broad processes. The Golgi apparatus was well developed. Many rosettes of free ribosomes were scattered throughout the cytoplasm, and the rough endoplasmic reticulum usually consisted of a few short cisternae. Small multilamellated bodies were common, but dense inclusion bodies were infrequent. The observations reported in this paper suggest: 1. that the nonneuronal cells which proliferate in a neuropil undergoing a mild denervation are morphologically closely related to microglia; 2. that in young adult animals these cells do not seem to have been previously involved in intense phagocytic activity; and 3. that the proliferating cells are present in the neuropil at the time of the denervation.
Animals↗
[A rapid method for staining semithin tissue sections embedded in araldite].
A new rapid method is proposed for staining of semithin sections. The method involves the treatment of sections with a methylene blue solution with a slight heating for 1-3 minutes. The method allows to receive polychromatic contrast preparations, to save time and reagents. It also permits to avoid restaining effect. The preparations can be preserved for a long time in plastic media (e.g. in polystyrene) for light microscopy. A comparative analysis of the staining methods used in the electron microscopic practice is given.
Animals↗
Assembly of the cricket cercal sensory system: genetic and epigenetic control.
The cercal sensory system of the cricket is being examined using anatomical, physiological, and computer simulation techniques in order to better understand the assembly of sensory systems. This particular sensory system is of interest because it functions like numerically more complex vertebrate sensory systems but offers, to the neuroscientist, the technical advantages of a small number of large identified neurons. Two aspects of sensory processing are being examined in this system; the spatial aspects of a stimulus that tell an animal where a target is in its environment, and the qualities of a stimulus that help the animal to identify the stimulus. The spatial aspects of a stimulus are analyzed by a topographic mapping of the animal's sensory environment. The feature extraction machinery for other aspects of the stimulus lacks any obvious anatomical order and is embedded within the topographic map. We are attempting to tease apart the genetic and the epigenetic components of the assembly process for this sensory system. Here we review our progress with emphasis on the epigenetic aspects of its assembly. We describe previously published work on plasticity as well as new experiments focussed on the role of neuronal activity in the assembly of this neural circuit. Finally, we briefly describe simulation experiments that are helping us understand the role of various forms of synaptic plasticity in the determination of receptive fields.
Animals↗
Quantitative investigation of scintillator intensification for light and electron microscope radioautography.
Several reports have indicated that scintillators enhance the intensity of radioautographic reactions. If this were the case, the use of scintillators would shorten the exposure time of radioautographs and the production of radioautographs would be accelerated. It was, therefore, decided to examine the effect of scintillators on the intensity of radioautographs in a quantitative manner over uniformly labeled test specimens. Reaction intensities with or without scintillators were compared using Kodak NTB2 emulsion for light microscopy and Ilford L4 and Sakura NR-H2 emulsion for electron microscopy. In addition, the background fog was quantitatively evaluated. No increase in the intensity of the radioautographs was observed under the following conditions: pretreatment of tissue sections with scintillator solutions before emulsion coating and exposure; treatment with scintillator solutions after emulsion coating but before exposure; treatment with scintillator solutions during exposure; contact with a plastic scintillator film throughout exposure. Heightened reaction intensity was obtained only when scintillators were applied during the histological processing of tissues. The impregnation of tissue blocks with PPO (2,5-Diphenyloxazole) and POPOP (1,4-bis-2-(5-phenyloxazolyl)-benzene) during dehydration, infiltration and Epon embedding produced a 20% increase of reaction intensity in light microscope radioautographs using NTB2 emulsion and a 75% increase in electron microscope radioautographs using L4 emulsion.
Animals↗
Imaging of 1.0-mm-diameter radiopaque markers with megavoltage X-rays: an improved online imaging system.
PURPOSE: To improve an online portal imaging system such that implanted cylindrical gold markers of small diameter (no more than 1.0 mm) can be visualized. These small markers would make the implantation procedure much less traumatic for the patient than the large markers (1.6 mm in diameter), which are usually used today to monitor prostate interfraction motion during radiation therapy. METHODS AND MATERIALS: Several changes have been made to a mirror-video based online imaging system to improve image quality. First, the conventional camera tube was replaced by an avalanche-multiplication-based video tube. This new camera tube has very high gain at the target such that the camera noise, which is one of the main causes of image degradation of online portal imaging systems, was overcome and effectively eliminated. Second, the conventional linear-accelerator (linac) target was replaced with a low atomic number (low-Z) target such that more diagnostic X-rays are present in the megavoltage X-ray beam. Third, the copper plate buildup layer for the phosphor screen was replaced by a thin plastic layer for detection of the diagnostic X-ray components in the beam generated by the low-Z target. RESULTS: Radiopaque fiducial gold markers of different sizes, i.e., 1.0 mm (diameter) x 5 mm (length) and 0.8 mm (diameter) x 3 mm (length), embedded in an Alderson Rando phantom, can be clearly seen on the images acquired with our improved system. These markers could not be seen on images obtained with any commercial system available in our clinic. CONCLUSION: This work demonstrates the visibility of small-diameter radiopaque markers with an improved online portal imaging system. These markers can be easily implanted into the prostate and used to monitor the interfraction motion of the prostate.
Equipment Design↗