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The juxtaglomerular apparatus in a human kidney with polar artery stenosis.

Juxtaglomerular apparatuses (JGA) of a human kidney with stenosis of a polar artery from a hypertensive 18-year-old male patient were studied qualitatively and quantitatively on 2 mu thick serial sections from plastic embedded renal tissue. 11 JGA from juxtamedullary and 9 JGA from subcapsular cortical zones were photographed serially, the copies taped together and the cells and relationships within the JGA studied. On the copies the lenght of contact between the different juxtaglomerular structures and the basement membrane of the macula densa was measured. Futhermore we calculated the areas of surface contact and the macula densa basal area. In the juxtamedullary JGA affected by the polar artery stenosis all Goormaghtigh cells were transformed into epitheloid cells and the Goormaghtigh cell field was significantly larger than in the JGA of the subcapsular cortex, which obviously had been exposed to the systemic hypertension. The macula densa basal area was significantly greater in the juxtamedullary JGA than in the subcapsular JGA, but neither of these differed significantly from the macula densa in the normal JGA reported previously.

Adolescent↗

Repeated TTS exposures in monkeys: alterations in hearing, cochlear structure, and single-unit thresholds.

The findings of a number of studies investigating the effects of excessive sound on hearing have indicated that the correspondence between behavioral, physiological, and histological measures of noise-induced hearing loss may be markedly dependent upon the sensitivity of the particular measure. Recent studies demonstrating significant changes in the responses of single auditory neurons following brief exposures to pure tones suggest that single-unit activity may be a sensitive indicator of physiological insult to the organ of Corti's sensory cells. In addition, the long-lasting nature of the changes in neural responsiveness suggests that each temporary threshold shift (TTS) episode may produce an increment of damage to the ear that eventually contributes to a measurable permanent threshold shift (PTS). A logical extension of this implication is the proposal that repeated episodes of TTS would first affect single-unit thresholds, and that such damage would eventually manifest itself as PTS. A test of this notion was performed by repeatedly exposing monkeys to short-lasting TTS sounds for many months. Behavioral thresholds were monitored using a reaction-time task before and after each inducement of TTS. Two subjects participated in exposure sessions for 18 months, while the remaining monkey was exposed to identical stimuli for 6 months. At the end of behavioral testing, the monkeys were prepared for chronic recording from single cells of the cochlear nucleus. Following the recording period, cochleas were prepared for examination as plastic-embedded whole mounts. Flat preparations of the cochlear duct were made and the position and extent of damage to the organ of Corti and myelinated nerve fibers were determined. No elevations in behavioral threshold were noted for the monkey receiving 6 months of sound-exposure experience, while for both subjects exposed for 18 months, a significant high-frequency hearing loss became apparent during the final months of exposure. For damaged ears, the thresholds of ipsilateral cochlear nucleus units were elevated for characteristic frequencies (CFs) corresponding to the frequency regions where behavioral thresholds were shifted. Thresholds for units with high-frequency CFs in the animal exposed for 6 months also demonstrated a loss in sensitivity. Histological examination of the cochleas of monkeys with permanent hearing losses revealed corresponding damage to the high-frequency region of the organ of Corti. The monkey exposed for 6 months, which demonstrated only elevated unit thresholds, also had high-frequency lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acoustic Stimulation↗

Morphologic study of Staphylococcus aureus L-form, reverting, and intermediate colonies in situ.

Staphylococcus aureus strains of bovine origin were induced to L-form by exposure to 100 U of penicillin in brain heart infusion broth supplemented with 5% NaCl, 5% sucrose, and 10% horse serum. L-forms were cultured on similarly supplemented brain heart infusion agar containing no antibiotic. Light and electron microscopic examination of plastic-embedded L-form colonies revealed a variety of morphologic types. The primary site of growth appeared to be the core area below the agar surface, consisting mainly of pleomorphic budding forms. At the surface, these forms gave rise to large spherules with a gradation from smaller to larger spherules toward the periphery of the colony. Some colonies progressed to reverting forms with the growth of bacterial cells containing cell wall. In addition to L-forms, intermediate colony forms were observed that lacked typical L-form morphology and progressed rapidly to the parent cell form on subculture to bovine blood agar. Description of these forms will be used in the search for similar morphologic types in vivo during antibiotic treatment of chronic S. aureus bovine mastitis.

Culture Media↗

Cytomegalovirus primary envelopment occurs at large infoldings of the inner nuclear membrane.

We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.

Animals↗

Detection of pseudorabies virus DNA in the inner ear of intranasally infected BALB/c mice with nucleic acid hybridization in situ.

Evidence for the pathogenicity of pseudorabies virus for the auditory and vestibular organs of experimentally infected mice is presented. We demonstrate viral genomes in cells of the peripheral sensory organs, the nerve structures, and the affected areas of the brain in single sections from an entire cranium of an adult mouse. The data were obtained by an in situ hybridization technique adapted for use with fixed, plastic-embedded materials. In contrast to conventional methods which use frozen sections, we were able to analyze cartilaginous and bony materials with high resolution.

Animals↗

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies.

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.

Antigens↗

Normal osteoid tissue.

The results of a histological study of normal osteoid tissue in man, the monkey, the dog, and the rat, using thin microtome sections of plastic-embedded undecalcified bone, are described. Osteoid tissue covers the entire bone surface, except for areas of active resorption, although the thickness of the layer of osteoid tissue varies at different sites and in different species of animals. The histological features of osteoid tissue, apart from its amount, are the same in the different species studied. Distinct bands or zones are recognizable in some layers of osteoid tissue, particularly those of greatest thickness, and their significance is discussed. Some of the histological features of the calcification front are described.

Animals↗

Enzyme histochemical analysis on cryostat sections of human bone marrow.

A recently developed procedure, that has been shown to be suitable for detailed immunohistological analysis, has been used to prepare cryostat sections of bone marrow to investigate whether enzyme-histochemical techniques are also feasible on such material. A selected group of enzymes, some of which are inhibited or destroyed in paraffin- or plastic-embedded samples, have been demonstrated. The morphological details obtained were satisfactory in the preparations. The enzymes were dipeptidyl(amino)peptidase IV (for T lymphocytes); tartrate-resistant acid phosphatase (for hairy cell leukaemia); acid phosphatase and non-specific esterase (for macrophages and monocytes); ATPase and 5'nucleotidase (for B lymphocytes); and peroxidase or chloroacetate esterase (for granulocytic cells). In these preparations strong enzyme activities were shown. In adjacent sections the immunological analysis of membrane markers could also be performed contributing to a comprehensive study of the normal and malignant bone marrow cells.

Bone Marrow↗

Morphometric characterisation of 52 B cell non-Hodgkin's lymphomas.

Fifty-two B cell non-Hodgkin's lymphomas, in which the diagnosis was based on immunological, cytochemical, and ultrastructural studies, were characterised by morphometry on plastic-embedded tissue sections. Parameters studied were: nuclear size, cytoplasmic area, cytoplasm to nucleus ratio, nuclear contour index, nucleolar size, location of nucleoli within the nucleus, as expressed by relative nucleolar eccentricity, and the mean number of nucleoli per nuclear cross-section. The results of the measurements and subsequent statistical analysis show that the different types of lymphoma can be distinguished from each other, even though the differences were small. Small cell lymphomas (chronic lymphocytic leukaemia, lymphoplasmacytoid and polymorphic immunocytoma, centrocytic, centroblastic/centrocytic and intermediate lymphocytic lymphomas) could be separated from each other by the standard error of mean (SEM) of nuclear area, the cytoplasm to nucleus ratio, and nuclear contour index. Large cell lymphomas (centroblastic and B immunoblastic lymphomas) could be differentiated with cytoplasm to nucleus ratio and nucleolar parameters (relative nucleolar eccentricity and number of nucleoli per nuclear cross-section). The morphometric parameters of lymphoblastic lymphomas fell in the range of the large cell lymphomas, but lower SEM indicate these lymphomas were more monomorphic. These morphometric data underline the concept of the Kiel classification and establish the usefulness of morphometry as an additional technique in diagnosis.

B-Lymphocytes↗

Biopsy specimen identification by detection of sex chromosomes: application of in situ hybridisation.

AIMS: To investigate the feasibility of non-radioactive in situ hybridisation (ISH) for the identification of sex-mismatched plastic embedded bone marrow biopsy specimens. METHODS: After a suspected accidental transposition of two glycol-methacrylate embedded bone marrow specimens, in situ hybridisation with sex chromosome specific probes was performed. RESULTS: Quantitative analysis of the hybridisation signals established unequivocably the origin of the specimens. CONCLUSIONS: ISH is feasible on GMA embedded bone marrow specimens, and can be used for the identification of accidentally transposed specimens provided that they are of sex-matched origin.

Bone Marrow↗

Experimental Campylobacter jejuni infection in the chicken: an animal model of axonal Guillain-Barré syndrome.

OBJECTIVE: To develop and characterise an animal model of paralytic neuropathy after Campylobacter jejuni infection. Campylobacter infection precedes development of many cases of Guillain-Barré syndrome and is particularly associated with cases having prominent axonal degeneration. Understanding the pathogenesis of Guillain-Barré syndrome after C jejuni infection has been slowed by the lack of animal models. METHODS: A spontaneous paralytic neuropathy is described that developed in chickens from the farms of four patients with Guillain-Barré syndrome. The production of paralytic neuropathy in chickens experimentally fed Campylobacter jejuni isolated from one of these patients is reported. The sciatic nerves of the spontaneously paralysed chickens were examined pathologically in teased fibres, in plastic embedded sections, and by electron microscopy. Two large groups of chickens were then fed cultures of a C jejuni (Penner type O:19) isolated from one of these patients. RESULTS: The chickens with spontaneous paralysis had pathologically noninflammatory neuropathy. Pathology in the sciatic nerves ranged from no detectable changes to severe Wallerian-like degeneration. In the experimentally inoculated groups, an average of 33% of the chickens became paralysed. The median time after inoculation to paralysis was 12 days. The lesions found in the first few days of paralysis included nodal lengthening and paranodal demyelination. In those animals that survived for several days after onset of weakness, the pathology was dominated by extensive Wallerian-like degeneration. Animals that survived for weeks with no clinically apparent neuropathy had paranodal remyelination in some teased nerve fibres, reflecting earlier paranodal demyelination. CONCLUSION: Experimental inoculation with C jejuni may provide a new model for understanding some forms of Guillain-Barré syndrome.

Animals↗

Recognition of 'trembler', a hypomyelinating condition in the Bernese mountain dog.

An account is given of a hypomyelinating condition, 'trembler', in the Bernese mountain dog. The condition is manifested clinically as a tremor of the limbs and head which becomes more intense with excitement or stress and which disappears with sleep. The tremor, which is first noticeable between two and eight weeks old, may persist throughout life but decline with age. Examination of plastic embedded tissue obtained post mortem from two, nine-week-old animals showed hypomyelination of the spinal cord. Preliminary examination of breeding data suggests that the condition may be inherited as an autosomal recessive.

Animals↗

The effect of chemical fixatives on cell walls of Bacillus subtilis.

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles. Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.

Acrolein↗

Dimensional growth of the rabbit eye.

The rabbit eye has been used experimentally in research in vision and ophthalmology. However, there is little information about postnatal growth. The purpose of this study was to determine growth patterns of the rabbit globe from birth to young adult status focusing in particular on the fibrous outer tunic of the globe, the sclera. Eyes of both sexes of New Zealand White rabbits were examined on postnatal days 1, 7, 14, 28, 42 and 56. Axial length, equatorial diameter and corneal diameter were measured using a digital caliper. Plastic-embedded tissue was used to quantify scleral thickness and cellular density at anterior, equatorial and posterior sites. Axial length increased 230% between postnatal days 1 and 56. The rate of growth was not linear and revealed two peaks. The first peak occurred between postnatal days 1-7, and the second between days 14-28. Eye opening occurred on days 9-11. Sex differences were seen, with the male eye being larger (p = 0.00001). Surface area measurements of sclera and cornea revealed that the sclera occupies approximately 5/6 of the outer surface of the globe and the cornea 1/6. Scleral thickness increased from day 1 to day 42 (70-155 microm at the equator) while fibroblast density was found to decrease (0.32 to 0.05 cells/microm(2)). The results suggested that eye opening precedes a unique increase in the rate of eye growth and visual processes may be playing a role in modulating the growth pattern between days 14 and 28.

Age Factors↗

Innervation of the dermatomes in the neck of the mouse.

The dorsal rami of the cervical and thoracic spinal nerves were investigated using both the in situ cholinesterase staining technique and cholinesterase staining on serial sections of plastic-embedded embryos. In most cases only the dorsal rami of the 2nd to 5th cervical spinal nerve possess cutaneous branches. The area innervated by the cutaneous branch of the dorsal ramus of the 5th spinal nerve borders on an area innervated by the cutaneous branch of the dorsal ramus of the 1st thoracic spinal nerve. The dorsal rami of the cervical spinal nerves 6-8 show no cutaneous branches. Therefore the gap in the series of the dorsal cutaneous branches is due only to the middle part of the nerves of the brachial plexus, which range from the 5th cervical nerve to the 1st thoracic nerve.

Animals↗

Fine structure of the epidermal basement membrane of the lip: applications of dithiothreitol separation and ultrathin frozen sectioning.

The fine structure of the epidermal basement membrane at the electron-microscopic level has already been defined. To obtain more details, two techniques, dithiothreitol separation and ultrathin frozen sectioning, were applied either alone or in combination. Negatively stained ultrathin sections showed a much thicker lamina densa than ordinary plastic-embedded sections. In the lamina lucida, bridging filaments and subbasal dense plates were observed in negative images. After the treatment with dithiothreitol, the lamina densa could be peeled off mechanically from the underlying dermis, and the anchoring fibrils were pulled off the dermis, preserving the connection with the lamina densa. With this specimen, bundles of anchoring fibrils were observed clearly and their lengths could be measured. Negatively stained ultrathin sections of dithiothreitol-separated specimens showed the fine structure of the lamina lucida. Bridging filaments in the lamina lucida were resolved by negative staining.

Animals↗

Glial-Schwann cell specialisations at the central-peripheral nervous system transition of a cyclostome: an ultrastructural study.

This study describes for the first time the central (CNS)-peripheral (PNS) nervous system transitional zone (TZ) of a member of the vertebrate class Agnatha, namely, the sea lamprey. It is concerned in particular with the glial tissue contribution to the TZ and the Schwann cell-CNS interface. Plastic-embedded specimens of spinal cord and nerve roots were examined using light and electron microscopy. At the TZ of each root a glial barrier, continuous with and similar in form to the surrounding glia limitans, stretches across the nerve bundle. In possessing such a barrier the lamprey TZ follows the general vertebrate pattern and differs from the Cephalochordata and invertebrates where a corresponding barrier is absent. The glial barrier is of similar thickness to the glia limitans generally, unlike in the mammal where it is thicker at the TZ than elsewhere. All lamprey axons are unmyelinated peripherally and most of them traverse the glial TZ barrier singly in individual tunnels. In this they resemble mammalian axons of similar calibre (which are, however, myelinated), but differ from unmyelinated mammalian axons, which generally traverse it in bundles. The lamprey TZ has specialisations not found in mammals. These include prominent, multiple interconnected strata of microfilament bundles which are continuous deeply with glial filament bundles and which lie under the surface plasmalemma of the glia limitans, to which they are connected by hemidesmosomes. Features of the mature lamprey TZ resemble those of developing mammalian TZs. For example, slender Schwann cell processes extend below the cord surface and become closely apposed to glial processes. In addition, together with the axon, the TZ glial processes and the basal lamina, they bound an extensive periaxonal network of spaces at the TZ, into which fine Schwann cell processes project. Accordingly, the networks could represent a primitive form of node gap.

Animals↗

Ultrastructural immunolabeling in renal diseases. Past, present and future expectations.

The use of immunoelectron microscopy in the evaluation of renal diseases represents a valuable addition to existing techniques. The ability to immunolabel immune-complex deposits for light- and heavy-chain determinants, and complement fractions virtually eliminates the need for traditional immunofluorescence. One of the main advantages of performing immunoelectron microscopy relates to the fact that immunologic events can be adequately correlated morphologically with the degree and type of tissue reactions in the various renal compartments. Immunopathogenetic mechanisms can be clearly evaluated with this technique. The need to submit specimens in different fixatives is eliminated and all diagnostic modalities can be performed on a single fragment of tissue. The examination of the thick sections prepared from plastic-embedded tissue for survey provides excellent material for interpretation of findings at the light-microscopic level. These sections can be stained with hematoxylin/eosin, trichrome, silver methenamine and PAS stains with excellent results. The time and resources needed for processing renal biopsies can be significantly reduced by utilizing the proposed methodology. Likewise, interpretation of results may benefit from utilization of the same fragment for all diagnostic modalities.

Complement System Proteins↗