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At least 973 records · Page 54Linked to original sources
PubMed2002

Biological staining: mechanisms and theory.

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.

Animals↗
PubMed1995

Method for embedding temporal bones of rats in methyl-methacrylate.

The commonly used method of preparing the temporal bone for light microscopy is a refinement of a basic formula that has been employed for a century. This process includes fixation, decalcification, neutralization, dehydration, embedding in celloidin, and hardening. The main disadvantage of this process is that decalcification is performed. This article describes a new method for preparing the temporal bone of rats for light microscopy. The main advantage of this new method is that no decalcification is involved, so that all bony elements are retained in their normal shape and location, and even retain some enzymatic activity. Other advantages are that the fixation is reversible and the process is short (approximately 2 weeks) and therefore relatively inexpensive. Our vast and positive experience with this technique has led us to report this method not in a specific experiment, but rather as a specific laboratory technique.

Animals↗
PubMed2001

Electron microscopic assessment of smeared or imprinted specimens of solid tumours.

The aim of this study was to develop a reliable method for preparation of smeared or imprinted cytology specimens for electron microscopic examination. Ten different solid tumours were studied. In each case one air-dried (Diff-Quik stained) and one alcohol-fixed (Pap stained) smear was prepared for diagnostic purposes. Simultaneously, a third smear or imprint was prepared for electron microscopy on a coverslip that was coated with poly-L-lysine and attached to a glass slide using double-sided adhesive tape. The smear or imprint was primary fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hours. The coverslip was removed from the slide, placed in a glass petri dish and processed for electron microscopy. The smear/imprint on the coverslip was embedded in resin on a silicon embedding mould and allowed to polymerise for 8-12 hours. The coverslip was removed using liquid nitrogen and the block sectioned using standard techniques as for a monolayer. The specimens collected for electron microscopy using this technique yielded sufficient material for assessment with excellent tissue preservation producing good ultrastructural detail. Focal mechanical damage was seen in some specimens but diagnostic areas were always found within the block. As the smear/imprint can be regarded as a monolayer, the processing time can be reduced compared with solid tissue specimens. This technique ensures that well-preserved tissue is available for electron microscopy even when the sample size is very limited.

Biopsy, Needle↗
PubMed2005

Use of surface affinity enrichment and cryo-embedding to prepare in vitro reconstituted mitotic chromosomes for EM tomography.

We present a novel preparation method for studies of in vitro reconstituted mitotic chromosomes from Xenopus laevis egg extracts. This method involves a gentle adsorption of chromosomes from the extracts using surface affinity enrichment, followed by plunge freezing, freeze-substitution and cryo-embedding before examination by EM tomography. For comparison, chromosomes were also prepared by a conventional method, which included immobilization of chromosomes in agarose and a room-temperature dehydration/embedding protocol. Three-dimensional reconstructions showed that samples prepared with the new method have a greater interconnectivity of sub-structural features and a higher compaction ratio together with an apparently less perturbed chromatin structure than those prepared using the conventional approach. The implications of the new method for the preparation of other difficult samples and additional application possibilities are discussed.

Animals↗
PubMed2000

Evaluation of guided bone regeneration in rabbit femur using collagen membranes.

The aim of the study was to evaluate the mechanical performance and the structure of neoformed bone around hydroxyapatite-coated titanium fixtures according to guided bone regeneration techniques. Ten hydroxyapatite-coated titanium fixtures were inserted in the femurs of five rabbits, in which a cortical defect was created and after the insertion of the fixture, covered with a resorbable membrane obtained from bovine Achilles tendon collagen Type I (A implant). In the same femur, a second fixture was inserted in similar cavities without application of the membrane (B implant). After 60 days, the animals were sacrificed, and block sections of the femoral bone containing the implants were embedded in polymethylmetacrylate and subjected to tensile shear-stress at break testing. After the detachment of the implants from the bone, their surfaces were examined with a scanning electron microscope. Tensile shear-stress values for A and B implant specimens were comparable to some extent, but the former had a lower performance. In this regard, scanning electron microscope observations showed that the neoformed cortical bone present cervically around implant A was much thicker than around implant B.

Absorbable Implants↗
PubMed1996

Cortical and total bone mineral content of the radius: accuracy of peripheral computed tomography.

The aim of the study was to evaluate the accuracy of an XCT 960 Stratec peripheral quantitative computed tomography (pQCT) device in assessing bone mineral content of the radius. We scanned 27 left forearm specimens excised from cadavers and focused on cortical bone mineral content (BMCc) at the junction of the middle and distal third and on total bone mineral content (BMCtot) at the distal end of the radius. Cylindrical specimens matched with those two sites were cut using a diamond circular saw, embedded in a polyester resin and subsequently submitted to two reference methods, nondestructive neutron activation analysis and flame atomic absorption spectrometry. Mineral contents measured by pQCT were closely correlated with those assessed by using the two reference methods, with correlation coefficients ranging from 0.862 to 0.960. The standard error of the estimate amounted 7-10% for the BMCc (junction of the middle and distal third), and 17-18% for the BMCtot (distal end). We conclude that pQCT is able to measure either cortical or total mineral content of the radius with a high degree of accuracy.

Aged↗
PubMedDate not supplied

Staining myelin and myelin-like degradation products in the spinal cords of chronic experimental allergic encephalomyelitis (Cr-EAE) rats using Sudan black B staining of glycol methacrylate-embedded material.

A high-resolution light-microscopical (HRLM) technique is described to visualize myelin, and macrophages containing degradation products of myelin, in the spinal cords of chronic relapsing experimental allergic encephalomyelitis (Cr-EAE) rats. This HRLM technique was developed to optimalize the correlation between nuclear magnetic resonance (NMR) characteristics and histopathological images in this well-established animal model for multiple sclerosis (MS). Spinal cords were fixed by perfusion with a combination of cacodylate-buffered glutaraldehyde and formaldehyde, post-fixed in Dalton's fixative (containing osmium tetroxide), rinsed in water, processed in ethanol, acetone, and embedded in glycol methacrylate resin (Technovit 7100/HistoResin). Semi-thin sections were stained with Sudan Black B and counterstained with Cresyl Fast Violet, resulting in black staining of myelin and its degradation products, with blue/violet staining of demyelinated axons and other tissue elements. These dyes were selected with the aid of a numerical model of staining, which took both access and lipophilicity into account. The staining procedure is simple and highly reproducible. The resulting images are contrast rich, and combine excellent morphology with a high degree of lipid retention.

Animals↗
PubMed1993

Ultrastructural characteristics of glial fibrillary acidic protein expression in epoxy resin-embedded human brain tumors.

Thirteen surgically removed, epoxy resin (Durcupan ACM or Epon 812)-embedded human brain tumors were examined for glial fibrillary acidic protein (GFAP) content in semithin and ultrathin sections with the immunogold-silver staining method. Mild aldehyde fixation and the hydrophobic resin embedding did not interfere with the antigenicity, since silver intensification of the immunogold marker provided excellent visualization of the reaction on both light microscopic and ultrastructural levels. The GFAP reaction was usually localized on the glial intermediate filament bundles, usually correlating well with the amount of filaments. The unstained filamental regions of two ependymomas might correspond to the vimentin expression revealed by double labeling in semithin sections. Occasional GFAP immunopositivity without filamental appearance was observed in one of the oligodendrogliomas, as patchy electron-dense cytoplasmic corpuscules, in Rosenthal fibers and in some mainly necrobiotic tumor cells, reflecting a possible connection with glial filaments.

Astrocytoma↗
PubMed2002

Optimization of glycol methacrylate embedding of large specimens in neurological research. Study of rat skull-brain specimens after implantation of polyester meshes.

Advances in neuroscience require better anatomical knowledge of neuronal architecture and structural details. Optimal embedding techniques are the basis for precise morphometric studies in section series as well as for the evaluation of tissue specimens or implants of differing hardness. There are very few methods for preparing large specimens by resin embedding, although resins such as polyethylene glycol (PEG) and methyl methacrylate (MMA) are presently in use. However, these methods have proven to be laborious and sometimes unsatisfactory for serial sectioning. While glycol methacrylate embedding (GMA) is suitable for smaller specimens, it results in inadequate infiltration and polymerization in blocks larger than 1 x 1 x 0.2 cm. We present an improved technique using GMA, which permits both standardized embedding of 4 x 2 x 2 cm blocks and preparation of section series. This method was developed for preserving skull-brain specimens from rats with polyester-mesh implants. The excellent preservation of cellular details allowed the assessment of local tissue reaction to foreign-body material in situ. Advantages of this method are: (1) No toxic catalysts or solvents are used (as opposed to MMA and current GMA processes); (2) Laborious routines in stretching and mounting of sections are not necessary (in contrast to PEG and MMA); (3) No deplastination is required before staining (in contrast to PEG and MMA); (4) Excellent morphologic preservation of various tissue is achieved.

Animals↗
PubMed1996

Glycocytochemistry of the mouse parotid gland. Investigation by lectin-gold techniques on Bioacryl and Lowicryl K4M embedded specimens.

In this study colloidal gold methodologies that allow the lectin affinity patterns in the mouse parotid gland to be studied by postembedding techniques were addressed. DBA, Con A, PNA, WGA and LTA lectins conjugated with gold particles or horseradish peroxidase for a direct or indirect technique of binding were used. Thin sections were obtained from tissue embedded in the acrylic hydrophilic resins Bioacryl and Lowicryl K4M. The most satisfactory results originated from sections infiltrated in Bioacryl and submitted to the indirect technique of binding. The mouse parotid gland appeared to be composed of polymorphous secretory granules differentially stained by lectins. The glycosylated components of acinar cells reacted intensely, and gold particles specified the ultrastructural distribution of lectin affinity sites reflecting the location of beta-galactose, alpha-N-acetylgalactosamine, beta-N-acetylglucosamine, alpha-D-mannose, and alpha-L-fucose in the electron-dense or electron-lucent areas of bizonal, mottled, and target granules.

Acrylic Resins↗
PubMed1999

Evidence for polar cytoplasm/nuage in rat oocytes.

In many organisms oocytes contain dark-staining material, termed nuage, that is concentrated at one pole of the oocyte cytoplasm and that influences the further development of the oocyte after fertilization. In mammalian oocytes, ultrastructural studies have detected small patches of nuage-like material, but thus far no nuage-rich zone of polar cytoplasm has been reported. Here, we report that when large sections of rat ovary embedded in methacrylate resin are stained with toluidine blue and surveyed, many oocytes contain a narrow, sharply defined, basophilic zone of polar cytoplasm that appears analogous to the polar cytoplasm of Xenopus and other non-mammalian species. This basophilic polar cytoplasm was common in multilaminar follicles and was not visible in smaller, primordial follicles. In one out of five oocytes stimulated with hCG to complete the first meiotic division, a relatively faint region of cortical basophilia was detectable. Further studies will be needed to ascertain if this nuage-like material has an influence upon the development of oocytes similar to that seen in non-mammalian species.

Animals↗
PubMed1982

Hairy cell leukemia with bone marrow hypoplasia.

This article reviews the authors' experience with hairy cell leukemia to determine the incidence of hypoplastic marrows in this disease and the utiity of embedding marrow biopsy specimens in plastic, which allows the sections to be stained for tartrate-resistant acid phosphatase (TRAP). It was found that the presence of hairy cells in the peripheral blood, marrow aspirate, or both established the diagnosis in eight of 11 patients; in the remaining three patients, the marrow biopsy specimen was necessary for definitive diagnosis. The bone marrow was markedly hypocellular in three patients, including one in whom the marrow morphologic results were essential for diagnosis. Embedding the marrow biopsy specimen in plastic and staining the sections for TRAP was noted to be a useful technique in establishing the diagnosis of hairy cell leukemia. In all cases in which this was performed, the authors found abundant TRAP-positive cells in the marrow biopsy specimen; even in those cases with markedly hypocellular marrows, this technique revealed the presence of hairy cells. In one patient, TRAP staining of the marrow biopsy specimen proved essential for definitive diagnosis. The authors concluded that the occurrence of marrow hypocellularity is not rare in patients with hairy cell leukemia and that TRAP-staining of plastic-embedded marrow biopsy specimens is a helpful and sometimes essential technique in diagnosing hairy cell leukemia.

Acid Phosphatase↗
PubMed1994

Evidence for a GABAergic interface between cortical afferents and brainstem projection neurons in the rat central extended amygdala.

The synaptic circuitry of the intrinsic GABAergic system of the central extended amygdala (CEA) in relation to efferent neurons and cortical afferents was examined in the present study. Neurons in the CEA projecting to the dorsal vagal complex and the parabrachial complex were identified by the retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Postembedding GABA-immunocytochemistry revealed that GABA-immunoreactive (GABA-IR) terminals formed largely symmetrical synaptic contacts with the perikarya and proximal dendritic processes of almost all WGA-HRP-labeled neurons in the CEA. To determine the relationship between cortical afferents and CEA GABAergic neurons, WGA-HRP was used to anterogradely label afferents from the insular cortex in combination with postembedding immunogold detection of GABA. Cortical afferents formed asymmetrical synaptic contacts predominantly on small dendrites and dendritic spines. Many of the dendrites postsynaptic to cortical terminals in the central nucleus were immunoreactive for GABA although only relatively few spines were GABA-IR. Combining pre-embedding GAD-immunocytochemistry with cortical lesions resulted in approximately 40% of degenerating terminals of insular cortical origin in the central nucleus in contact with small, GAD-IR dendrites and spines. The present results demonstrate that the neurons providing the major CEA outputs to the brainstem receive an extensive GABAergic innervation, strongly supporting our proposal that CEA efferent neurons are under strong tonic inhibition by intrinsic GABAergic neurons. Further, our finding that the major cortical input to the central nucleus preferentially innervates intrinsic GABAergic neurons suggests that these neurons in the CEA may serve as an interface between the principal inputs and outputs of this forebrain region.

Amygdala↗
PubMedDate not supplied

A polychromatic staining method for epoxy embedded tissue: a new combination of methylene blue and basic fuchsine for light microscopy.

A simple and rapid method is described for staining semithin sections of material embedded in epoxy resin for observing tissues prior to transmission electron microscopy. The method is suitable for tissue fixed with a glutaraldehyde-formaldehyde mixture and postfixed in osmium tetroxide. No etching or oxidizing procedures are necessary. Sections 0.5-0.8 microm thick are dried onto a slide and stained with either 0.75% methylene blue and 0.25% azure B or 0.5% methylene blue and 0.5% azure II in 0.5% aqueous borax and heated over a flame for 8-10 sec. The slides are rinsed with water, then stained the same way with 0.1% basic fuchsine in 5% aqueous ethanol. Cytoplasm stains blue; nuclei darker blue; collagen, mucus and elastin pink to red; fat and intracellular lipid droplets gray-green.

Animals↗
PubMed1995

Bromodeoxyuridine and iododeoxyuridine double immunostaining for epoxy resin sections.

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.

Animals↗
PubMed1992

Ultrastructural localization of human papilloma virus by nonradioactive in situ hybridization on tissue of human cervical intraepithelial neoplasia.

BACKGROUND: A nonradioactive in situ hybridization was developed to localize human papilloma virus (HPV) at the ultrastructural level. EXPERIMENTAL DESIGN: Cervical biopsies from human uterine cervices clinically suspicious of condyloma were embedded in Lowicryl K4M at low temperature. Postembedding in situ hybridization was performed with DNA probes specific for HPV types 6/11, 16, and 18. The hybrids were detected by anti-horseradish peroxidase antibodies conjugated with 10 nm colloidal gold particles. RESULTS: Localization for HPV 16 and 18 both was to intranuclear and cytoplasmic sites. Cytoplasmic detected HPV signals were between masses of intermediate filaments and in vacuoles; other organelles were devoid of positive signal. Within the nucleus the precise localization of the viral nucleic acid was episomal, vacuolar, and chromosomal. In situ hybridization with plasmid control DNA confirmed the specificity of the HPV positive signals. CONCLUSIONS: This study helps define the subcellular compartmentalization of HPV DNA in infected human cells.

Epithelium↗
PubMed1997

Immunophenotype of hairy-cell leukaemia after cold polymerization of methyl-methacrylate embeddings from 50 diagnostic bone marrow biopsies.

Hairy-cell leukaemia may be difficult to diagnose in bone marrow biopsies, especially in the early stages or in its residum after complete clinical remission. To consider the impact of published data on immunophenotyping hairy-cell leukaemias, a total of 50 diagnostic biopsies were systematically analysed with a panel of eight antibodies and compared with cases of chronic lymphatic leukaemia (CLL), 20 follicular centre lymphomas, 20 lympho-plasmacytoid immunocytomas, 10 small-cell T-cell non-Hodgkin lymphomas and 20 cases of benign nodular lymphatic hyperplasia. The panel of eight antibodies comprised DBA44, CD45, CD20, CD45R, CD45RO, CD43 and the CD68 antibodies KP1 and Ki-M1P. The hairy-cell leukaemias were staged histologically into four categories of bone marrow infiltration. DBA44 reacted positively in 47/50 cases. CD45 and the B-cell markers CD20 and CD45R reacted in 49/50 and 43/50 cases, respectively. One CD68 marker, KP1, was positive in 38/50 cases but the other-Ki-M1P-only in 1/50 cases. Chronic lymphatic leukaemia cases, the other B-cell NHLs and lymphatic hyperplasias showed strong positivity for CD20 and CD45R, but only the immunocytomas reacted with DBA44 in 7/20 cases. The T-cell NHLs and hyperplasias showed a strong positivity for the T-cell markers CD45RO and CD43. The CD68-marker Ki-M1P revealed a high specificity since it was negative in all NHLs and positive only in one hairy-cell leukaemia. Methyl-methacrylate embedding of bone marrow biopsies under cold polymerization produces a high quality of histo- and cytomorphology, resulting in greater diagnostic reliability and the detection of low-stage infiltration of hairy-cell leukaemia. DBA44 appears as a highly specific antibody to mark hairy-cells since only immunocytomas reacted positively in a few cases. A small panel of antibodies including DBA44. CD20, CD45R and Ki-M1P may serve to distinguish small-cell. NHL from hairy-cell leukaemia even at an early stage or when there are minimal residual tumour cells.

Adult↗
PubMed1992

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.

Animals↗