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[A histopathological study of bone marrow in acute myeloid leukemia. Bone marrow biopsy changes before chemotherapy and comparison with bone marrow smears].

Histopathological changes of pretreatment bone marrow biopsy from 42 cases with acute myeloid leukemia (AML) were described. A considerable difference was shown between the results of aspirate smears and the findings of plastic embedded biopsy sections, particularly in bone marrow cellularity such as infiltration of inflammatory cells and presence of residual hemopoietic cells, qualitative and quantitative abnormalities of megakaryocytes suggestive of myelodysplastic features were more accurately assessed in the sections of marrow biopsy than in the aspirate smears. In three cases there was considerable infiltration of maturing but dysplastic granulocytic cells and erythroid precursors in the sections, but not in the aspirate smears. Our study shows that plastic embedded biopsy sections provide more information than aspiration smears for the diagnosis of AML.

Adolescent↗

Calibration of [123I]iodine-labeled tissue standards for autoradiographic studies.

Autoradiographic images can be analyzed with computer-assisted microdensitometry relative to radioactive reference standards to provide quantitative measurements of regional radioactivity concentrations. [123I]Iodine containing sections of brain paste have been calibrated relative to plastic-embedded tritium(3H) and 125Iodine standards. For exposure times of 8, 12 and 24 h, plastic-embedded standards covered a range concentrations of [123I]iodine from 1000 to 100,000 dpm/mg wet weight. Iodine-123 radioactivity was linear with section thickness from 5 to 30 microns. These studies confirmed the feasibility of using commercially available longer-lived reference standards to provide [123I]iodine equivalent values in absolute units of dpm (or microCi) per mg tissue.

Animals↗

Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions.

Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications for in situ detection, identification and localization of microorganisms in veterinary as well as in human medicine.

Animals↗

In situ localization of enzymes and mucin in normal rat colon embedded in plastic.

Several enzymes were investigated histochemically in the colons of normal male F344 rats in order to understand the function of different types of cells in this tissue. Serial methacrylate-embedded sections (2-4 microns) allowed the precise localizations of several enzymes including acid phosphatase, alkaline phosphatase, gamma-glutamyl transpeptidase, N-acetyl-beta-D-glucosaminidase (hexosaminidase), alpha-naphthyl butyrate esterase and 5'-nucleotidase. Sites reactive with periodic acid-Schiff were also localized. Gradients of enzyme activity were observed between caecum and rectum and/or from the luminal surfaces to the bases of the crypts for hexosaminidase, esterase and gamma-glutamyl transpeptidase. To our knowledge this is the first histochemical demonstration of gamma-glutamyl transpeptidase in normal rat colonic epithelial cells. The utilization of the methacrylate-embedding technique has revealed previously undescribed gradients of enzyme activity and has allowed the localization of enzyme activities not previously reported in normal rat colonic mucosa.

Animals↗

Ciprofloxacin overdose: acute renal failure with prominent apoptotic changes.

Acute renal failure due to ciprofloxacin has been well described. The previously reported cases have been consistent both clinically and pathologically with tubulointerstitial nephritis (TIN). We report a case of ciprofloxacin overdose leading to acute renal failure characterized by acute tubular necrosis (ATN). The outstanding features in the patient's renal biopsy were those of distal nephron apoptosis, best seen in plastic-embedded sections and electron microscopy. This report shows that the distal nephron can be singularly involved in acute renal failure and that electron microscopy or plastic-embedded sections may be necessary to define its involvement.

Acute Kidney Injury↗

Bone core biopsies. Plastic or paraffin?

Plastic embedding of bone core biopsy specimens has been promoted as providing superior morphology, primarily because semi-thin sections can thereby be cut at 1-2 mu. The major disadvantages of plastic embedding are that it increases the technical load, is more expensive, and potentially has its own intrinsic problems, including difficulties in performing special stains and immunoperoxidase studies. In order to investigate the possibility that semi-thin paraffin sections may provide similar morphological results without the intrinsic disadvantages of plastic sections, we examined 45 bone core biopsy specimens that were sufficiently large to process one half in plastic and the other half in paraffin following decalcification. Both were cut at 1-2 mu. Although many plastic sections appear esthetically more pleasing, semi-thin paraffin sections of very high quality can also be obtained routinely. Additional advantages of paraffin sections were the ability to perform peroxidase studies, lower cost, less technologist time, and avoidance of problems occasionally arising with plastic, such as difficulties with impregnation or problems with polymerization. Peroxidase studies were particularly useful in patients with possible myeloma that was not overt on hematoxylin-and-eosin section and in confirming the presence or source of metastatic carcinoma. We therefore recommend the use of semi-thin (1-2 mu) paraffin sections for routine examination of bone core biopsy specimens.

Biopsy↗

Scanning electron microscopy of plasma etched implant specimens.

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.

Animals↗

Immunohistological staining of antigens on semithin sections of specimens embedded in plastic (GMA-Quetol 523).

Glycol methacrylate-Quetol 523, introduced by Kushida (1977) for combined light and electronmicroscopy studies at low magnification, also permits application of immunofluorescence methods to semithin sections. To recover the antigenicity of proteins fixed with formaldehyde, abrupt dehydration before embedding and subsequent treatment of the semithin sections with protease were essential. Post-staining with suitable histological stains allows exact correlation of antigen localization with tissue structure.

Acrylic Resins↗

Measurement of the repeat period of myelin sheath using ultrathin frozen sections.

The myelin sheath of peripheral nerves was observed by transmission electron microscopy (TEM) using plastic-embedded sections and ultrathin frozen sections. Repeat distances of myelin sheaths were measured in high-powered electron micrographs. The ultrathin frozen sections showed a longer repeat distance than the plastic-embedded sections. The ultrathin frozen sections were thought to contain fewer artefacts, as they had not been subject to dehydration and embedding. It is known that broken myelin sheaths are often observed under conventional TEM. It is thought that these procedures cause contraction and partial destruction of the myelin sheath.

Animals↗

Clinicopathologic correlation of an untreated macular hole and a macular hole treated by vitrectomy, transforming growth factor-beta 2, and gas tamponade.

PURPOSE: To study the clinicopathologic features of an untreated macular hole in the right eye and a macular hole in the left eye treated by vitrectomy, application of transforming growth factor-beta 2, and gas tamponade. METHODS: The patient, a 73-year-old man with bilateral macular holes, was studied clinically before and after surgical treatment of the macular hole in his left eye. The patient's eyes were obtained postmortem and serially step-sectioned through the macula and optic nerve head for electron microscopy. RESULTS: Examination of 1-micron thick plastic-embedded sections through the macula of the right eye disclosed a 0.6-mm macular hole with rounded gliotic margins, a thin epiretinal membrane, and parafoveal cystic changes. Examination of 1-micron thick plastic-embedded sections through the macula of the left eye disclosed a 0.25-mm defect in the fovea, which was bridged by glial cells. The glial cells were continuous with a thin hypocellular epiretinal membrane without contraction features on both sides of the defect. The ultrastructural features of the glial cells were consistent with Mueller cells. CONCLUSIONS: Treatment of a macular hole with vitrectomy, transforming growth factor-beta 2, and gas tamponade was followed by complete closure of the macular hole by Mueller cell proliferation.

Aged↗

Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements.

Comparative morphological examination and elemental analysis were carried out in structural compartments of sections of skeletal muscles. These had been prepared either by conventional plastic embedding technique or by various methods of cryo-ultramicrotomy. The analyses were performed in a Philips EM 301 with an Edax energy-dispersive X-ray spectrometer. Spectra obtained from sections of plastic-embedded muscle depended on the reagents used for fixation and staining and were absent if these were omitted. Brief fixation with glutaraldehyde resulted in gross ionic changes, and sectioning of frozen material with trough liquid led to extraction of elements. Sections cut from unfixed and frozen muscle without trough liquid showed numerous peaks. (Mg, P, S, Cl, K, Ca). In the superficial parts of the fibres of freeze-dried sections reproducible spectral differences were found between different structures. Thus, rapid freezing of unfixed tissue, dry cutting in the frozen state, and freeze-drying should be the procedure of choice if data on diffusible ions are desired.

Animals↗

Ultrastructural detection of herpes-type virions by negative staining in oral hairy leukoplakia.

Hairy leukoplakia (HL) is a newly recognized virus-associated lesion of oral mucosa that occurs in persons infected with human immunodeficiency virus. Studies have demonstrated Epstein-Barr virus within epithelial cells of HL. The authors examined 12 cases of HL by transmission electron microscopy to compare the use of a negative staining technique versus routine plastic embedment for the detection of viruses. Herpes-type virions were identified by both methods in 11 cases. One case had negative results for viruses by both methods. Negative staining is a simple and rapid technique that compares favorably with plastic embedment in the detection of herpes-type virions in HL.

Acquired Immunodeficiency Syndrome↗

Neural architecture of the rat medial geniculate body.

The rat medial geniculate body was subdivided using Nissl preparations to establish nuclear boundaries, with Golgi-Cox impregnations to identify projection and local circuit neurons, and in fiber stained material to delineate the fiber tracts and their distribution. Three divisions were recognized (ventral, dorsal and medial): the first two had subdivisions. The ventral division had lateral and medial parts. The main cell type had bushy tufted dendrites which, with the afferent axons, formed fibrodendritic laminae oriented from dorso-lateral to ventro-medial; such laminae were not as regular medially, in the ovoid nucleus. The dorsal division contained several nuclei (dorsal superficial, dorsal, deep dorsal, suprageniculate, and ventrolateral) and neurons with radiating or bushy dendrites; the nuclear subdivisions differed in the concentration of one cell type or another, and in packing density. A laminar organization was present only in the dorsal superficial nucleus. Medial division neurons were heterogeneous in size and shape, ranging from tiny cells to magnocellular neurons; the various cell types intermingled. so that no further subdivision could be made. This parcellation scheme was consistent with, and supported by, the findings from plastic embedded or fiber stained material. There were very few small neurons with locally ramifying axons and which could perform an intrinsic role like that of Golgi type II cells. Their rarity was consistent with the small number of such profiles in plastic embedded or Nissl material and the few GABAergic medial geniculate body neurons seen in prior immunocytochemical work. While similar neuronal types and nuclear subdivisions are recognized in the rat and cat, there may be major interspecific differences with regard to interneuronal organization in the auditory thalamus whose functional correlates are unknown.

Animals↗

Orientation staining for the demonstration of Mycobacterium leprae in semithin sections.

In Spurr-embedded biopsies for ultrastructural examinations, Mycobacterium leprae hardly differed from the surrounding tissues using the staining technique of Richardson. Also, the usual histological staining methods of Ziehl-Neelsen and Fite did not achieve positive results for the determination of M. leprae. Therefore, we applied the methylene blue-borax and basic fuchsin technique for the demonstration of the bacilli in plastic-embedded tissue of 11 patients suffering from Hansen's disease. In every patient the diagnosis was confirmed by histological examination of skin biopsies. Portions of the biopsies of nine patients were then fixed in glutaraldehyde and osmium tetroxide and embedded according to Spurr. In the other two cases, the material was first fixed in 10% Formalin and embedded in paraffin. After cutting 3-micron sections for routine histological examination, the remaining material was prepared adequately for ultrastructural examination. Using the methylene blue-borax and basic fuchsin technique, the semithin sections of plastic-embedded material presented a considerably more differentiated picture than other comparable methods. M. leprae located in foamy cells or in the tissue stained violet. These findings were corroborated in subsequent electron-microscopic examinations. The semithin sections thus prepared allow, through the clear demonstration of the microorganisms, a precise demarcation of the ultrathin area.

Biopsy↗

Podocyte expression of MHC class I and II and intercellular adhesion molecule-1 (ICAM-1) in experimental pauci-immune crescentic glomerulonephritis.

We examined immunopathological changes of podocytes in vivo which, based on in vitro studies, are thought to be relevant for the pathogenesis of renal diseases. We investigated the alterations of podocytes in local inflammation in a recently developed model of pauci-immune necrotizing crescentic glomerulonephritis (NCGN) in the rat. Frozen and plastic embedded kidney sections at different time points of the disease were incubated with antibodies directed to MHC class I, MHC class II, ICAM-1 and to relevant cytokines. Strong glomerular expression of MHC class I, II and ICAM-1 was found within 4 days, and plastic embedded sections clearly demonstrated increased cell membrane staining of podocytes. Increased glomerular interferon-gamma (IFN-gamma) was detected within 24 h of induction of NCGN, and IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) were found from day 4. The potency of these cytokines to induce adhesion molecules on podocytes was investigated on rat glomerular epithelial cells in vitro. By using FACS analysis and electron microscopical techniques, we found that the in vivo expression of MHC class I, II and ICAM-1 by podocytes could in vitro be simulated by IFN-gamma. IFN-alpha weakly induced MHC class I, while IL-1 beta and TNF-alpha were ineffective. We hypothesize that podocytes in this in vivo model are important to maintain the local inflammatory process in the glomerulus by expression of relevant adhesion molecules and MHC molecules upon stimulation with specific cytokines.

Animals↗