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Ultrastructure and three-dimensional organization of the telangiectases of hereditary hemorrhagic telangiectasia.

We studied 10 cutaneous telangiectatic lesions of hereditary hemorrhagic telangiectasia (HHT), ranging in size from pinpoint to 2 mm, by light and electron microscopy. Four representative lesions were reconstructed by computer from serial 1- or 2-mm plastic embedded sections. The earliest clinically detectable lesion of HHT is a focal dilatation of postcapillary venules, which continue to enlarge and eventually connect with dilated arterioles through capillaries. As the vascular lesion increases in size, the capillary segments disappear and a direct arterio-venous communication is formed. This entire sequence of morphologic events is associated with a perivascular mononuclear cell infiltrate in which the majority of cells are lymphocytes and the minority are monocytes/macrophages by ultrastructure. Comparison of these findings with the telangiectatic mats of scleroderma and cherry angiomas revealed that the former, previously shown to be composed of dilated postcapillary venules, are also associated with perivascular infiltrates, but the latter, which are produced by capillary loop aneurysms, are not.

Adult↗

The extension technique: a new method of demonstrating initial lymph vessels in excised human skin.

The initial lymph vessels, the lymph capillaries and precollectors, are difficult to detect in normal skin using routine histological methods. A method is described in which specimens of the skin are fixed in an expanded condition. In 1.05 microns thick plastic-embedded sections the dilated lymph vessels of the skin can be demonstrated. The dermal lymph vessel network can be seen following the subepidermal injection of a coloured solution. The endothelial cells and subendothelial fibrous network can be examined using electron microscopy.

Histological Techniques↗

Bone marrow diagnosis in lymphoproliferative disorders: comparison of results obtained from conventional histomorphology and immunohistology.

In this study we have investigated 313 bone marrow biopsies from 280 patients with lymphoproliferative disorders. Trephines were sectioned transversely to obtain one cylinder for cryostat sectioning and immunostaining and a second for histomorphological evaluation using a plastic-embedding technique. The results obtained by histomorphological and immunohistological evaluation were compared for their contribution to staging and classification. Using both techniques, bone marrow involvement was seen in 3/43 (7.0%) biopsies from patients with Hodgkin's disease and in 193/270 (71.5%) cases with non-Hodgkin's lymphoma, including multiple myeloma and acute lymphocytic leukaemia. Immunohistology proved superior in detecting minimal mainly interstitial bone marrow infiltration in 15 leukaemia/lymphoma cases. Biopsies showing infiltration with both methods (n = 157) were re-examined for classification of lymphomatous infiltrates. Whereas immunohistology did not provide additional information in cases with Hodgkin's disease and myeloma, this method was crucial for establishing the definitive diagnosis in a number of cases with acute lymphocytic leukaemia and non-Hodgkin's lymphoma. In all of six leukaemia cases, in which no or inadequate material was available for immunophenotyping of cell suspensions, immunohistology clearly defined the subtype. In the 140 cases of non-Hodgkin's lymphoma the majority of cases (76.4%) were identically classified. In some cases, with important prognostic and therapeutic implications, immunohistology alone provided the definitive diagnosis: T-cell lymphoma (n = 2), hairy cell leukaemia (n = 2) and centrocytic non-Hodgkin's lymphoma (n = 3). Bone marrow immunohistology is, therefore, an important supplement for classical lymphoma/leukaemia diagnosis. The differences observed between histomorphology and immunohistology emphasize the importance of lymph node biopsy in lymphoma classification.

Biomarkers↗

Restricted lesions after testicular biopsies in young and adult rats.

In order to evaluate the possible harmful effects of surgical removal of a testicular biopsy, adult and immature rats were subjected to unilateral testicular biopsy and were studied 2-4 months later. One group of adult rats were sham-operated. Perfusion-fixed, plastic-embedded specimens of the testes were examined by light microscopy. No morphological differences were found between rats that were immature and those that were adult at the time of biopsy. The lesions observed were focal and occurred only in the vicinity of the site of biopsy. Only about 0.5% (range 0.01-4.5) of the testis was affected. No morphological signs of any immune reaction were observed. It is suggested that the lesions are caused mainly by interference with local blood flow, and to a minor extent by disruption of the flow of seminiferous tubule fluid.

Age Factors↗

High-resolution autoradiography of 3H-ouabain binding in salt transporting epithelia.

The distribution of salt transporting sites in epithelia is a problem of special interest to electrolyte metabolism. Autoradiography of freeze-dried plastic embedded tissue was used to localize 3H-ouabain in rabbit small intestine and renal medulla and gills of killifish. Resolution approaching theoretical limits for tritium at the light microscope level was achieved. The results of several tests are presented which demonstrate the resolution of the method and the specificity of the observed ouabain binding patterns. A large uptake of ouabain was found along the basolateral membranes of the enterocyte, thick ascending tubules and chloride cells (fish gill).

Animals↗

High-resolution autoradiography as a tool for the localization of nucleic acid synthesis and distribution in the mammalian cell nucleus.

Several examples of the application of high resolution autoradiography to the study of nucleic acid distribution in ultrathin sections of fixed and plastic embedded or frozen material are presented. Newly-synthesized DNA, labelled by very short pulses of 3H-thymidine is found to be localized throughout the nucleus. In the blastomeres of early mouse embryos developing after fertilization by 3H-thymidine-labelled spermatozoa, the labelled paternal DNA is distributed non-homogeneously in the nucleus. Finally some results obtained by using a cytochemical enzymatic technique for visualization of DNA directly in ultrathin sections of fixed and embedded cells are demonstrated. Concerning the distribution of RNA, perichromatin localization of rapidly transcribed RNA is described in different cell systems. This label can be attributed to perichromatin fibrils. In cells labelled for longer periods of time, sometimes followed by prolonged postincubations, a residual label is always found over the nucleus. Clusters of interchromatin granules are usually only weakly labelled or unlabelled. These structures probably contain a limited amount of rather slowly labelled RNA. The present results are discussed in the context of some biochemical evidence and of the data described by other investigators.

Animals↗

The use of PIPES buffer in the fixation of mammalian and marine tissues for electron microscopy.

PIPES, an organic based buffer, was used in the preparation of a variety of marine and mammalian tissues for electron microscopic study. The ultrastructural results demonstrate that this buffer renders superior ultrastructural details to those obtained with some of the more common inorganic buffers especially when long fixation times are required. Furthermore, this buffer system does not appear to contribute extraneous anions or cations to the tissues and thus permits accurate elemental determinations to be performed on thin sections of the plastic embedded specimens by means of energy dispersive X-ray analysis.

Animals↗

A simple method for maintaining relative positions of separate tissue elements during processing for electron microscopy.

Molten (328 K) 20% gelatin is used as a 'glue' to hold together separate tissue elements or tissue elements that may be separated when cutting small blocks of tissue for plastic embedding. Standard aldehyde and osmium fixation, dehydration and epoxy embedding are compatible with this as is semi-thin sectioning for light microscopy or thin sectioning for electron microscopy.

Animals↗

Morphometric studies on mouse bone using a computer-based image-analysis system.

The morphological structure of the ilium, femur, third lumbar vertebra and a central caudal vertebra of the female CBA mouse has been studied using 5 micrometer thick, plastic embedded, transverse and longitudinal sections. The sections were analysed on a Quantimet 720, system 30, image analyser connected on-line to a PDP11 computer. Separate endosteal and periosteal surface to volume ratios were calculated for each position of sampling in each bone. For this calculation the anisotrophy of the bone was estimated from measurements of mean chord lengths in longitudinal sections of the bone using a new analytical technique. Chord length distributions in transverse sections of bone were also measured and the relevance of such measurements to the study of morphological changes in the bone and its included marrow are briefly discussed.

Animals↗

Vitreous cryo-sectioning of cells facilitated by a micromanipulator.

Sectioning vitrified cells and tissues for cryo-electron microscopy is more challenging than room-temperature sectioning of plastic-embedded samples. As the sample must be kept very cold (<-130 degrees C) and because there is no liquid upon which the sections can float as they are cut, transferring the sections from the knife edge to a grid is one of the more difficult steps in the process. We employed a micromanipulator to hold and control the cryo-sections as they come off the knife. This allows slower cutting speeds than are typically used in vitreous cryo-sectioning and contributes to better control during cutting, which facilitates repeatable placement of a ribbon of sections onto a grid. The ribbon is kept under tension during the entire cutting process, which may decrease folding and/or compression, features that are inherent to vitreous sections. Furthermore, the added control afforded by this technique makes it easier for multiple ribbons to be placed on a single grid, thereby increasing the number of sections that can be examined and imaged during a microscopy session. It even allows for serial cryo-electron microscopy. As such, this approach is an advance in the cryo-microtomy of vitreous sections.

Cryoelectron Microscopy↗

Computed tomographic anatomy of the canine temporal bone.

This study aims to identify and describe structures of the temporal bone in normal dogs that can be depicted using computed tomography (CT). Five normal dogs of different breeds and both sexes ranging from 0.5 to 8 years were used as the study material. CT images of 2 mm thickness without abnormalities in the temporal bone were reviewed and analysed in bone and soft tissue settings. All images were compared with corresponding E 12 plastic-embedded cross sections of 3 mm thickness, which were acquired from anatomic cross sections of frozen beagles. The sections were provided from the Department of Pathobiology (Institute of Anatomy). CT is able to image all relevant bony structures within the temporal bone. Concerning soft tissue structures, CT allows their localization by using bony markers, i.e. the internal acustic meatus for the cranial nerves VII and VIII or the canals for the internal carotid artery and the temporal sinus. The provided reference images are meant to improve orientation within the complex anatomy of the temporal bone. On condition that slice thickness is equal to or smaller than 2 mm and bony setting is supplied, CT is able to visualize all relevant structures within the temporal bone. However, in clinical settings the combined look on air, liquids and bone as well as the contrast enhancement of soft tissues might prefer magnetic resonance imaging to CT.

Animals↗

Comparative morphology of the tapetum lucidum (among selected species).

OBJECTIVES: The phenomenon of 'eye-shine' is seen in a variety of animal species, and is generally thought to be related to the presence of an intraocular reflecting structure, the tapetum lucidum. The tapetum lucidum is a biologic reflector system that is a common feature in the eyes of vertebrates. It normally functions to provide the light-sensitive retinal cells with a second opportunity for photon-photoreceptor stimulation, thereby enhancing visual sensitivity at low light levels. The tapetum lucidum is presented here according to a classification based on the location, as well as the composition, of this reflective layer. Finally, the physical and chemical properties, as well as the origins of the different tapeta lucida, are discussed and compared. METHODS: The anatomic and biochemical aspects of the tapetum lucidum in various vertebrates are examined. Morphologic observations were made from paraffin and plastic embedded specimens. Specimens were treated with traditional stains and observed by light and transmission electron microscopy. RESULTS: Some species (primates, squirrels, birds, red kangaroo and pig) do not have this structure and they usually are diurnal animals. In vertebrates, the tapetum lucidum exhibits diverse structure, organization and composition. Therefore, the retinal tapetum (teleosts, crocodilians, marsupials, fruit bat), the choroidal guanine tapetum (elasmobranchs), the choroidal tapetum cellulosum (carnivores, rodents, cetacea), and the choroidal tapetum fibrosum (cow, sheep, goat, horse) are described. CONCLUSIONS: The tapetum lucidum represents a remarkable example of neural cell and tissue specialization as an adaptation to a dim light environment and, despite these differences, all tapetal variants act to increase retinal sensitivity by reflecting light back through the photoreceptor layer. These variations regarding both its location and structure, as well as the choice of reflective material, may represent selective visual adaptations associated with their feeding behavior, in response to the use of specific wavelengths and amount of reflectance required.

Animals↗

New applications of electron microscopy techniques in dermatopathology.

The application of immunostaining techniques to electron microscopy specimens has led to a renewal of interest in electron microscopy in biological research in general, as well as in dermatopathology. Refinements in the preparative procedures have made easier the immunolocalization of antigens both in chemically-fixed and frozen unfixed tissues, embedded in plastic and sectioned. Application of these methods has led to the demonstration of the bullous pemphigoid antigen inside basal keratinocytes. HMB-45 antigen has been found to be present in premelanosomes. Recently, Factor XIIIa has been localized not only in dermal dendrocytes but also in endothelial cells and mast cells.

Antigens, Neoplasm↗

Comparative bone morphometric analysis of mandibles and iliac crests.

A comparative analysis of bone mass in the mandible and the iliac crest has been carried out in autopsy specimens from 30 subjects aged 23-84 years. Microradiograms of standardized 100-micrometer-thick sections of the undemineralized plastic embedded material from the two locations were used for quantitation. The following measurements were carried out by electronic point-counting: 1) the percentage of cortical bone mass in the subperiosteal and subendosteal layers, 2) mean cortical width, and 3) the percentage of trabecular bone mass in the iliac crest. Positive correlations were found in the expressions of bone mass within the mandible; the values were significantly higher in the 200-micrometer-thick subperiosteal layer than in the remaining part of the mandibular cortex. Only a poor or no correlation was found in the expressions of bone mass from the two sites. The investigation, therefore, shows that bone mass and cortical width in the mandible cannot be predicted from a biopsy from the iliac crest. This finding may partly be due to different age-dependent functional changes of the two bones.

Adult↗

Tissue distribution of epimucosally applied 3H DNFB: an autoradiographic study.

The distribution of 3H-labeled 2,4-dinitrofluorobenzene (DNFB) has been autoradiographically investigated in buccal mucosa after topical application to sensitized or nonsensitized rats. The rats were killed between 3 min and 24 h after challenge. No significant labeling pattern differences were found between plastic embedded and frozen sections. Surface epithelium was heavily labeled and labeled cells were observed in the lamina propria shortly (3-6 min) after application. These cells were clearly dendritic. Specific accumulation of hapten in epithelial Langerhans cells (LC) could not be clearly demonstrated. The connective tissue labeling gradually diminished and at 24 h post-elicitation, remaining label could be detected only in the epithelial surface layers.

Administration, Cutaneous↗

Close microtopographical relationships between sympathetic nerve terminals and bulbous process endings of pinealocytes in the pineal gland of the Mongolian gerbil.

Previous studies have shown that pinealocytes of the gerbil pineal gland exhibit processes that form terminal swellings filled with abundant electron-lucent microvesicles. The membrane of these presumptive secretory microvesicles is known to contain synaptophysin, a major integral glycoprotein of neuronal synaptic vesicles. The present study was conducted to evaluate the microtopographical relationships between the vesicle-rich process swellings and intra-pineal nerve terminals. For this purpose, both nerve terminals and pinealocyte process endings were visualized immunohistochemically in the same semi-thin sections of plastic-embedded gerbil pineals, using antibodies directed against synaptophysin. This approach consistently revealed close spatial associations of punctate immunopositive nerve endings with intensity stained bulbous process terminals of pinealocytes in or near the perivascular spaces. The light-microscopic observations of intimate neuronal-pinealocytic relationships were corroborated at the electron-microscopic level. Perivascular varicosities with ultrastructural features characteristic of sympathetic nerve terminals were frequently juxtaposed to vesicle-filled process endings of pinealocytes. Analysis of serial thin sections showed that multiple point-to-point contacts are encountered between noradrenergic nerve terminals and pinealocytic process swellings. Our morphological findings imply that bulbous process terminals, at least in the gerbil pineal gland, are major targets for the neuronal control of the secretory activity of pinealocytes.

Animals↗

Rat and gerbil pinealocytes contain the synaptosomal-associated protein 25 (SNAP-25).

It has recently been established that the neuroendocrine pinealocytes of mammals contain several synaptic membrane proteins that are involved in the regulation of vesicle trafficking in the nerve terminal. In the present study, we have conducted immunoblot and immunocytochemical analyses to demonstrate that another key component of the presynaptic plasmalemma, i.e., protein SNAP-25 (synaptosomal-associated protein 25 kDa), can be detected in pinealocytes. Immunostaining of serial semi-thin sections of plastic-embedded rat and gerbil pineals with monoclonal SNAP-25 antibodies showed that SNAP-25 was present in pinealocytes of both species. We proved its coexpression with other synaptic membrane proteins (synaptophysin, synaptotagmin I, synaptobrevin II, and syntaxin I) at the single cell level. Thus, pinealocytes obviously are endowed with the major proteins that are thought to regulate the targeting and exocytosis of secretory vesicles, in particular of synaptic-like microvesicles.

Animals↗

Histomorphometric analysis of normal bone from the iliac crest.

Bone specimens from a standardized area of the iliac crest were obtained at autopsy in 105 individuals after sudden unexpected death and at biopsy in 30 living volunteers. Seven micron thick sections of the plastic embedded undecalcified material were produced and stained. The amount of cancellous and cortical bone and the parameters attached to description of bone remodelling were determined by point counting and simple measurements, in order to establish normal mean values and range in a Danish population. A decrease in the amount of bone, trabecular as well as cortical, was found with increasing age in both males and females. The extent, volume and width of osteoid seams and the osteoclastic resorption were found to be age-independent. The osteoclastic resorption in cortical bone in males decreased, however, with increasing age. The reported values are of the greatest importance for the use of histomorphometric analysis of bone biopsies as a diagnostic tool in metabolic bone diseases. It is stressed, however, that supplementary information of the dynamic aspects of the bone remodelling would be necessary for the understanding of the pathogenesis of bone changes. Such information might be obtained by using tetracyline double labelling.

Adolescent↗