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In situ analysis of microvascular pericytes in hypertensive rat brains.

We used immunofluorescence microscopy and isoactin-specific antibodies to characterize the pattern and prevalence of pericytes within the brain microcirculation. Blood pressures of normotensive, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were measured prior to sacrifice and pressure-perfusion fixation. WKY and SHR brains were subdivided into ten major regions prior to ultracryomicrotomy. Sections 0.3-0.5 micron wide were treated with 10-40 micrograms/ml affinity-purified antibodies to the muscle and non-muscle actin isoforms. These localization studies show that there are four times the number of pericyte-rich capillaries in the SHR motor cortex compared to WKY counterparts (59.9 vs. 15.3%). In contrast, the sensory cortex of both rat strains is deficient in muscle actin staining surrounding the capillaries. The most striking difference in pericyte presence and muscle actin antibody staining between the SHR and WKY was observed in the tegmentum of the brainstem. There is nearly a one-to-one coincidence observed in pericyte and capillary profiles present within thin, frozen sections of the SHR midbrain. SHR pons capillaries were also pericyte-enriched. WKY analyses of plastic embedded thin sections confirmed the presence of pericytes and their filament-enriched processes encircling the capillaries of the hypertensive brains. These results suggest that pericytes may play important roles in hypertension and cerebrovascular disease processes.

Actins↗

Structural study of the frog Rana temporaria larval stomach.

The gastric wall of Rana temporaria tadpoles consists of a well-developed mucosa and thin muscular and serosa layers. Three cellular types--mucous, ciliated and endocrine cells--make up the lining epithelium. Different types of endocrine cells exist. Argyrophylic endocrine cells can be recognized in semithin sections of plastic-embedded material while non-argyrophylic endocrine cells can only be identified under the electron microscope. Glands are composed mainly of well-differentiated oxyntic cells and, occasionally, scarce endocrine cells. Oxyntic cells show abundant mitochondria and smooth endoplasmic reticulum, but do not contain zymogen granules as do those present in adults. Secretory canaliculi with microvilli are also well-developed. The lamina propria contains numerous vascular sinuses and nerve bundles which innervate the endothelium and some endocrine cells. The neuroendocrine regulation of frog gastric functions seems therefore to have developed in young tadpoles. Nerve fibers also innervate the muscular propria, which is composed of a single layer of smooth muscle cells. Underlying the muscle, connective fibers and a flattened layer of mesothelial cells make up the serosa. In summary, the structure of the frog larval stomach shows a well-differentiated histological pattern, especially referring to surface epithelium and glands. Some of the histological traits will also be present in adult frogs while others are characteristic of the tadpole's stage.

Animals↗

Method for ultrastructural studies of the intact tissue-metal interface.

Samples were prepared for ultrastructural studies of the intact interface between metallic implants and tissue by transmission electron microscopy. The method is based on plastic embedding of implant and tissue and subsequent removal of the bulk metal by electrochemical dissolution (electropolishing), to facilitate preparation of ultrathin sections for transmission electron microscopy. Surface sensitive spectroscopy (Auger electron microscopy and X-ray photoemission spectroscopy) and transmission electron microscopy EDX results show that the method produces samples with an intact interface, containing the implant surface oxide and the adjacent tissue. Examples of application of the method on titanium, zirconium and aluminium implants in soft tissue are given.

Aluminum↗

Histological reaction to titanium alloy and hydroxyapatite particles in the rabbit tibia.

The interfacial membrane harvested from failed joint replacements contains particulate debris from the materials used for the implant. To define the tissue response to particulate titanium alloy and hydroxyapatite (HA) alone, 16 mature New Zealand white rabbits were divided into 2 groups of 8 rabbits. Using sterile technique, a drill hole was placed anteromedially in the tibia, 1 cm distal to the knee joint bilaterally. The marrow was scooped out and 0.25 mg of either titanium alloy particles or HA particles were inserted in the right tibia. The titanium alloy particles had a diameter averaging 4.0 +/- 4.4 microns (mean +/- standard deviation) and an aspect ratio (the ratio of the maximum length divided by the maximum width) of 1.84. The HA particles had a diameter of 4.4 +/- 3.3 microns and an aspect ratio of 1.76. The left leg was prepared in a similar fashion, but no biomaterial was implanted. The animals were killed after 16 wk. The harvested tibiae were processed with decalcification and the plastic-embedded sections were subjected to histomorphological analysis. Black titanium alloy particles were present within the bone marrow fat between haematopoietic cells, and within scattered macrophages. The surrounding bone appeared to be unaffected. Within the spongiosa, the HA particles were surrounded by small numbers of mononuclear histiocytes or encased within a shell of new appositional bone. Where HA deposits were exposed to the endosteal aspect of bone, there was scalloping of the surface of the HA in a pattern suggestive of resorption or dissolution of the HA particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloys↗

Immunohistological assessment of bone marrow biopsies from patients with hairy cell leukemia: changes following treatment with alpha-2-interferon and deoxycoformycin.

Forty-six bone marrow biopsies from twelve hairy cell leukemia (HCL) patients, treated with either interferon(IFN)-alpha-2 (n = 8) or 2'deoxycoformycin(DCF) (n = 4), were examined using cryostat sections and an immunoperoxidase technique. Using this sensitive method we were able to demonstrate residual hairy cell (HC) infiltration in five cases, in which evaluation with conventional staining techniques on plastic embedded biopsies revealed complete remission. The amount of HCs in these five samples ranged from 1 to 7% (mean: 3%) of bone marrow cells. Consecutive biopsies in individual HCL patients revealed no changes of the immunological phenotype (CD19, CD22, CD25, CD10, CD11c, FMC7, HLA-DR, surface immunoglobulins) during IFN and DCF treatment. Within the infiltrated bone marrow a considerable number of "reactive" T lymphocytes was identified with prevalence of the T-helper (CD4+) subtype in untreated cases, whereas T-suppressor/cytotoxic (CD8+) cells were within the normal range. IFN treatment resulted in a reduction of CD4+ T lymphocytes (p less than 0.02). Minor alterations of CD8+ T lymphocytes and NK cells (HNK-1 + lymphoid cells) were found in bone marrow during IFN treatment. In DCF-treated patients bone marrow T lymphocytes were markedly reduced below the values of normal bone marrow. This DCF-induced T-cell depression might be related to the clinical observation of persistent cellular immune dysfunctions in HCL patients despite a DCF-induced remission.

Adult↗

Distribution of VIM-2 and SSEA-1 glycoconjugate epitopes among human leukocytes and leukemia cells.

Anti-SSEA-1 which binds to glycoconjugates with a Gal beta 1-4(fuc alpha 1-3)GlcNAc epitope and VIM-2 which binds to gangliosides with a NeuAc alpha 2-3GlcNAc beta-4(FUC alpha 1-3) GlcNAc beta 1-3Gal-epitope were used to determine the expression of their corresponding carbohydrate antigens in human leukocytes and leukemia cells. Expression of these antigens was evaluated by immunohistochemical staining of plastic embedded sections of bone marrow or isolated cells, and by immunostaining of isolated glycosphingolipids separated by thin layer chromatography. The expression of both antigens was restricted to normal and leukemic myeloid cells. A range of positive immunohistochemical staining was found among normal marrow myeloid precursors, with myeloblasts giving weaker staining than more mature cells (promyelocytes, myelocytes, metamyelocytes). A similar trend was observed with leukemia cell lines, in that the myeloblastic cell line KG1 was weakly stained compared to the partially differentiated cell line HL-60. Immunohistochemical staining of marrows from acute leukemia patients showed that the VIM-2 antigen is more strongly expressed than the SSEA-1 antigen. Interestingly, both antibodies stained AMMoL cells more intensely than AML cells. Granulocytes from marrows of chronic myelogenous leukemia (CML) patients were intensely stained by both antibodies, whereas lymphocytic leukemias (acute lymphocytic, chronic lymphocytic and hairy cell marrows) were negative. Thus, although both antigens are restricted to myeloid cells there are differences in the level of expression depending on the level of cell maturity. Immunostaining of glycosphingolipids isolated from myeloid cells demonstrated that the SSEA-1 epitope is carried by several neutral glycosphingolipids and that the VIM-2 epitope is carried by three or more gangliosides. Major SSEA-1 glycosphingolipids, with seven to more than ten monosaccharides, are expressed by all myeloid cells regardless of the level of maturity, although quantitative differences are apparent in different patient samples. Two strongly immunoreactive VIM-2 gangliosides with ten and twelve monosaccharides, respectively were found in myeloid cells. The ratio of these two gangliosides varied dramatically, with greater amounts of the more complex ganglioside being present in most cell samples. Normal neutrophils and CML cells had much greater quantities of the VIM-2 gangliosides than acute leukemia cells. This observation correlates with our earlier findings that: (1) acute leukemia cells have less total ganglioside than granulocytes and (2) acute leukemia cells have a predominance of short chain gangliosides (i.e. less than five monosaccharide units). Finally, both CML cells and normal neutrophils express a shorter chain VIM-2 ganglioside, which was not detected in acute myelogenous leukemia cells.

Antigens, Surface↗

Cell cycle and clinical characteristics of patients with acute myeloid leukemia and myelodysplasia whose biopsies are reactive with anti-factor VIII antibody. A Leukemia Intergroup Study.

Presence of megakaryocytic cells in patients with myeloid disorders were investigated by staining plastic embedded biopsy sections with an anti-Factor VIII antibody (AFA). Two hundred and fifty cases were studied, 207 of whom had acute myeloid leukemia (AML) while 43 had myelodysplastic syndromes (MDS). Abnormal clusters of AFA positive cells indicating multilineage disease were identified in 17% with primary AML (30/175), 38% with secondary AML (12/32) and 42% cases of MDS (18/43). Biological characteristics of these 60 AFA positive cases were investigated. No unique differences in cell cycle characteristics following bromodeoxyuridine (BrdU) were identified. We confirm several recent reports that the incidence of multilineage involvement in AML is substantial.

Adult↗

Morphologic classification of the myelodysplastic syndromes (MDS): combined utilization of bone marrow aspirates and trephine biopsies.

In a retrospective and prospective follow-up study from 1975 to 1991, bone marrow biopsies, aspirates and clinical features of 495 patients with MDS were investigated. Sections of undecalcified plastic embedded biopsies and smears of bone marrow aspirates were stained according to Giemsa. Bone marrows with MDS were characterized by three main categories of morphologic alterations: (1) cellular abnormalities, (2) architectural disorganization in the bone marrow and (3) stromal changes; the combined use of aspirates and trephine biopsies enabled a more reliable and accurate diagnosis of MDS than either one alone. The bone marrow findings fell into one of 7 subtypes, with the frequencies and median survivals in brackets: (1) MDS sideroblastic (19%, 62 months), (2) MDS megaloblastoid (13%, 56 months), (3) MDS proliferative (22%, 31 months), (4) MDS blastic (15%, 9 months), (5) MDS hypoplastic (15%, 26 months), (6) MDS fibrotic (6%, 29 months), and (7) MDS inflammatory (10%, 42 months). In follow-up studies patients with secondary MDS were excluded and the prognosis and subsequent evolution for each of the morphologic subtypes were evaluated. The conclusion is drawn that aspirates and trephine biopsies are complementary procedures and both are required for diagnosis, classification and decisions on current treatment modalities of patients with MDS.

Adolescent↗

Ultrastructural study of unencapsulated vertebrate mechanoreceptor terminals facilitated by double staining and resectioning of thick plastic sections.

A set of techniques for localization of unencapsulated sensory receptor terminals in plastic-embedded tissue is described. The tissue is pinned flat in fixative and flat-embedded in a small amount of medium-soft Epon. Pieces of the specimen are remounted so that tangential thick sections (2-3 micron) can be cut. These sections are stained with a mixture of 15 ml of 0.5% toluidine blue, 1% sodium borate; 10 ml of 2% p-phenylenediamine, and 5 ml of acetone. Stained sections are examined with a light microscope and brown-stained sensory receptor regions are located. Sections to be examined in the electron microscope are remounted by inverting a capsule of medium-hard Epon over them, and polymerized blocks are removed from the slide after heating it. Thin sections cut from remounted thick sections are stained with uranyl acetate and lead citrate. This procedure has been used successfully to locate aortic baroreceptors and very small apparent nerve endings in the atrium of the rat as well as stretch receptors in dog trachealis muscle. All of these receptor endings stain brown and are surrounded by light blue collagen and darker blue Schwann cells.

Animals↗

In vivo staining of the mammalian retina by means of a simple fluorescent method.

Intraocular injections of microliter volumes of a solution containing the fluorescent stain Fluoro-Gold produces an intense and long-lasting in vivo stain of the cells forming the neural retina of rats and mice. The label is incorporated by the neuro-retinal cells, while other intraocular structures such as the lens, iris, ciliary processes and even the optic nerve head exclude it. The fluorophore can be easily demonstrated under epi-illumination by means of commonly used fluorescence filter combinations. The stain has proven to be compatible with cryostat sectioning and with plastic embedding. Furthermore, electron dense lysosomal and lamellar bodies in retinal neurons which have incorporated the stain may provide a means for identification of Fluoro-Gold labeled cells at the electron microscopic level which is consistent with previous results. Intraocular injection of Fluoro-Gold fills the need for a reliable, simple, and robust in vivo and in toto stain of the neural retina.

Animals↗

A method of in situ hybridization combined with immunocytochemistry, histochemistry, and tract tracing to characterize the mRNA expressing cell types in heterogeneous neuronal populations.

A rapid, sensitive, non-isotopic in situ hybridization histochemistry protocol is presented to study the expression of mRNA at the single cell level in anatomically complex structures of the mammalian central nervous system. The protocol uses digoxigenin-UTP-labeled riboprobes, freefloating sections, and alkaline phosphatase and horseradish peroxidase detection. Modifications have been introduced which preserve the integrity of marker molecules, and as such enable the simultaneous identification and characterization of CNS cell types by tract tracing, histochemical, and immunocytochemical detection of intra- and extracellular markers. All pretreatments that enhance probe penetration have been omitted without substantial loss in sensitivity. The protocol has been successfully extended to vibratome sections with subsequent plastic-embedding and semithin sectioning, which considerably broadens the general applicability of this fast and easy ISHH method.

Animals↗

Sensitive detection of nucleic acids and protein of human papillomavirus type 6 in respiratory and genital tract papillomata.

We have developed a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in biopsy specimens of genital and respiratory tract lesions by using in situ hybridization and immunoperoxidase assays on sections of plastic-embedded tissue. This modified in situ hybridization technique, using ultrathin sections and strand-specific 3H-labelled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution. This modified immunocytochemistry provides better sensitivity when compared to previous methods using paraffin-embedded materials. In respiratory tract lesions, immunoperoxidase assay detected only a few capsid antigen positive cells, while in the genital tract lesions, there were more capsid antigen positive cells. Southern transfer analyses and in situ hybridizations demonstrated the presence of more viral nucleic acids in genital tract papillomata than respiratory tract papillomata. Epithelial cells throughout the papillomata were infected by HPV-6 as evidenced by positive hybridization, with more viral DNA present in superficial cells. Our results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. Using stand-specific probes synthesized from subgenomic fragments of the HPV-6 genome in conjunction with nuclease digestions, we were able to demonstrate that HPV-6 transcripts specific to open reading frames (ORFs) E6, E7, E1, L1, and L2 occur in maturing superficial cells. In contrast, transcripts specific to ORFs E1, E2, E4, E5a, and E5b could be detected throughout the whole of the epithelium with more signals noted at the basal cell areas. In addition, the distribution of HPV-6 nucleic acids and protein in a carcinoma in situ of the larynx was analyzed. In comparison to benign respiratory tract papillomata, more viral DNA was found in the malignant lesion, but the pattern and distribution of transcription and capsid antigen was similar.

Antigens, Viral↗

Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord.

Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities.

Animals↗

Back-scattered electron imaging of skeletal tissues.

The use of solid-state back-scattered electron (BSE) detectors in the scanning electron microscopic study of skeletal tissues has been investigated. To minimize the topographic element in the image, flat samples and a ring detector configuration with the sample at normal incidence to the beam and the detector are used. Very flat samples are prepared by diamond micromilling or diamond polishing plastic-embedded tissue. Density discrimination in the image is so good that different density phases within mineralized bone can be imaged. For unembedded spongy bone, cut surfaces can be discriminated from natural surfaces by a topographic contrast mechanism. BSE imaging also presents advantages for unembedded samples with rough topography, such as anorganic preparations of the mineralization zone in cartilage, which give rise to severe charging problems with conventional secondary electron imaging.

Animals↗

Identification of the mineralization front: comparison of a modified toluidine blue stain with tetracycline fluorescence.

A modified toluidine blue method for identification of the mineralization front at the zone of demarcation between bone and osteoid in undecalcified, plastic-embedded sections of bone is described. The intensity of staining is increased both by increasing the pH of the solution and by increasing the duration of staining. The method is reproducible, since measurements of the extent of mineralization front on two nonadjacent sections from the same biopsy in 20 cases had a correlation coefficient of 0.98. The identification of the mineralization front by toluidine blue agrees closely with identification by means of in vivo tetracycline uptake, with a correlation coefficient of 0.97 between measurements of its extent using the two methods on adjacent sections from the same biopsy. It is likely that both toluidine blue staining at pH 6.5 and tetracycline uptake depend on some property of the most recently deposited bone mineral. With either method we find low values for the extent of mineralization front as a fraction of osteoid surface in many patients with osteoporosis. The uniformly normal values for this quantity in osteoporosis reported by other investigators may reflect different criteria for distinguishing osteoid-covered from quiescent bone surfaces.

Biopsy↗

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy.

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.

Animals↗

Lung injury and repair: DNA synthesis following 1,1-dichloroethylene.

Injury and cellular proliferation in the lung were examined following administration of 1,1-dichloroethylene (1,1-DCE) or vinylidene chloride. C57BL/6 male mice were treated orally with 200 mg/kg of 1,1-DCE prior to a single pulse of tritiated thymidine [( 3H]TdR). Necrosis and exfoliation of Clara cells of bronchiolar epithelium were evident by 1 day after chemical administration, and increased in severity by 2 days. A regenerative response was observed at 3 days after 1,1-DCE administration, and by 7 days the epithelium was substantially restored. At 30 days after 1,1-DCE, re-epithelization was achieved and areas devoid of epithelium were not observed. Changes in cellular proliferation were calculated from measurements of [3H]TdR incorporation into total pulmonary DNA. Activity of [3H]TdR was significantly inhibited at 1 day after chemical administration and thereafter increased: a peak of synthesis occurred between 3 and 5 days. At 7 days after 1,1-DCE administration, incorporation of [3H]TdR decreased to levels that were not significantly different from those of control animals. Autoradiographic examination of 0.5 micron thick plastic-embedded lung sections showed that [3H]TdR was incorporated into the DNA of bronchiolar epithelial cells, macrophages, interstitial, endothelial and Type II alveolar cells. However, the majority of the label was taken up by the nonciliated bronchiolar epithelial cells. The increased [3H]TdR incorporation into whole lung correlated with repopulation of bronchioles which was observed following injury. The results demonstrated that 1,1-DCE-induced damage to Clara cells of the bronchiolar epithelium was severe and rapid; re-epithelization was achieved in a relatively short time whereas differentiation was a prolonged process.

Animals↗

Olfactory mitral cell integrity after freeze-fixation.

The condition of freeze-fixed mitral cells from the main olfactory bulb of the rat brain was found to depend on the rat at which the tissue is frozen: at very slow rates, mitral cells swell and rupture; at faster rates, cell integrity is preserved. Cooling the tissue to just above its freezing point before rapid freeze-fixation helps to maintain cell integrity at depths well below the surface. When freeze-fixation must be used, as in the 2-deoxyglucose technique, rapid freezing rates should be used to minimize diffusion of isotope and increase autoradiographic resolution. After rapid freezing, better cell integrity was produced by freeze-drying and plastic embedding than by cryostat sectioning and thaw-mounting.

Animals↗