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At least 325 records · Page 18Linked to original sources
PubMed1992

Quantitative analysis of neurons and glial cells in the rat somatosensory cortex, with special reference to GABAergic neurons and parvalbumin-containing neurons.

The number of neuronal and glial cells in the rat somatosensory cortex (barrel area) has been estimated by a stereological method, the disector, using pairs of toluidine blue-stained, plastic-embedded 0.5-microns-thick sections, 1.5 microns distant from each other. Chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent semithin sections. Thus we were able to analyze quantitatively number, distribution, and proportion of five cell types: (1) gamma-aminobutyric acid-(GABA)-negative neurons; (2) GABA-like immunoreactive (GABA-LIR) neurons; (3) a specific calcium-binding protein parvalbumin-immunoreactive (PV-IR) neurons, a subpopulation of GABA-LIR neurons; (4) S-100 beta-LIR glial cells (astrocytes); and (5) S-100 beta-negative glial cells (oligodendrocytes and microglia). The densities of total cells, glial cells, and neurons in the rat somatosensory cortex were 85.4 +/- 10(3)/mm3, 30.5 x 10(3)/mm3, and 54.9 x 10(3)/mm3, respectively. Of all neurons 25% and 14% were GABA-LIR and PV-IR, respectively; all PV-IR neurons are GABA-LIR, and thus about 54% of GABA-LIR neurons are PV-positive. The number of total cells under a unit surface area of 1 mm2 through the thickness of the somatosensory cortex was 171.6 x 10(3); the number of neurons and glial cells were 110.2 x 10(3) and 61.4 x 10(3), respectively. There were 27.7 x 10(3) GABA-LIR neurons and 15.0 x 10(3) and 12.7 x 10(3) PV-IR neurons and PV-negative GABA-LIR neurons, respectively. The laminar distribution of each group of cells shows prominent differences, indicating that the cellular composition was different from layer to layer. The density of GABA-LIR neurons was highest in layer IV. The numerical density of PV-IR neurons was 2-4 times higher in layer IV than in layers II/III, V, and VI, whereas that of PV-negative GABA-LIR neurons was almost constant throughout the layers.

Animals↗
PubMed1994

Quantitative analysis of GABA-like-immunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method, the disector.

The numerical density of neurons in the CA1 region of the rat dorsal hippocampus has been estimated by a stereological method, the disector, using pairs of video images of toluidine blue-stained, plastic-embedded, 0.5-microns-thick sections, 3 microns distant from each other. The chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent, semithin sections using antibodies against gamma-aminobutyric acid (GABA) and a specific calcium-binding protein, parvalbumin (PV). The density of neurons in the CA1 region was 35.2 x 10(3)/mm3; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 x 10(3)/mm3, 272.4 x 10(3)/mm3, and 1.9 x 10(3)/mm3, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 x 10(3)/mm3 and 1.1 x 10(3)/mm3, respectively, which were 5.8% and 3.2% of all neurons, respectively. In the CA1 region only about 60% of PV-positive neurons were GABA-LIR. However, taking the previous observation into consideration that almost all hippocampal PV-positive neurons were immunoreactive for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), neurons that were immunoreactive to either GABA or PV or both (GABA+ and/or PV+ neurons) were regarded as a better representative of GABAergic neurons in this region; thus, the numerical density of these GABA+ and/or PV+ neurons was 2.5 x 10(3)/mm3 and they were 7.0% of all neurons in the CA1 region. Lamellar analysis showed that the numerical densities of GABA+ and/or PV+, GABA-LIR, and PV-IR neurons were highest in the SP, where they were 8.2 x 10(3)/mm3, 6.2 x 10(3)/mm3, and 5.4 x 10(3)/mm3, respectively. The results of the present study indicate that the proportions of GABAergic neurons and a subpopulation of them, PV-containing GABAergic neurons, to other presumable non-GABAergic neurons are far smaller in the CA1 region of the hippocampus than in several neocortical regions previously reported.

Animals↗
PubMed1982

Trimming selected areas of embedments for electron microscopy--dead simple.

Precision trimming of specific areas of plastic embedments can be accomplished without having to find the specimen detail in the block face itself. The desired area is located in a thick section of the pretrimmed block. The position of the area in the section is evaluated using an ocular micrometer. The block is then mechanically trimmed in the ultramicrotome in such a way that the amount of plastic removed from each of its sides is determined from the magnitude of the knife advance. The procedure can be used in connection with inclined blocks if the microtome head can be rotated behind the specimen orientation arc.

Microscopy, Electron↗
PubMed1987

Rapid preparation of fresh-frozen undecalcified bone for histological and histochemical analysis.

An increasing need to be able to relate hard tissue morphology to its histochemistry, and the lack of an adequate methodology, prompted this investigation of polyvinylpyrrolidones of varying chain length as supporting films for the cryomicrotomy of fresh undecalcified cortical and trabecular bone. We developed a method that parallels histological procedures but is biochemically inert and functions at -25 degrees C. Using an LKB heavy-duty cryomicrotome, large numbers of serial sections suitable for histochemistry, immunocytochemistry, or rapid histology can be prepared with ease. Sections from mouse skull, ox vertebra, and human iliac crest illustrate the versatility of the method, and have been examined for the preservation of general morphology and of histochemical activity of those enzymes most prominent in remodeling. The procedure does not preclude the subsequent application of standard hard-tissue embedding methods to the remaining material. Comparison with plastic-embedded bone shows not only close similarities in the structural preservation of the frozen tissue, making possible histomorphometry, but also shows some interesting staining differences. These include the absence of differentiation of the calcification front with toluidine blue stain in fresh frozen specimens, and the presence in frozen osteoid tissue of specific methylene blue metachromasia missing from plastic preparations.

Acid Phosphatase↗
PubMed1987

Leu-M1 antigen as a marker of acute nonlymphoid leukemia.

The monoclonal antibody Leu-M1 was originally described as a marker of myeloid/monocytic cells and their precursors. Its usefulness as a myeloid marker in bone marrow specimens was further supported by a limited number of studies using flow cytometry and cell suspensions. Recent reports on its utility in paraffin sections for the diagnosis of acute leukemia have not shown it to be an effective marker of myeloid differentiation. To further evaluate the diagnostic usefulness of Leu-M1 in acute leukemia, we investigated the immunoreactivity of Leu-M1 antigen in a series of 100 plastic-embedded bone marrow biopsies from patients with acute leukemia and compared these results to those obtained in a series of 30 paraffin-embedded specimens. We also investigated the usefulness of neuraminidase pretreatment of sections to enhance the sensitivity of Leu-M1. In plastic sections, we were able to demonstrate positive Leu-M1 staining in 48 of 50 (96%) cases of acute nonlymphoid leukemia (ANL) (FAB classification M1-M5) and 0 of 50 cases of acute lymphocytic leukemia (ALL). Comparatively, Leu-M1 stained positively in 10 of 20 (50%) cases of ANL and 0 of 10 ALL embedded in paraffin. In both plastic and paraffin sections, pretreatment with neuraminidase enhanced the sensitivity of Leu-M1 reactivity, but markedly decreased its specificity. Our study suggests Leu-M1 is both a reliable marker of myelomonocytic differentiation and a useful diagnostic tool in the differentiation of ANL from ALL in plastic sections, and confirms its limited usefulness in paraffin-embedded material.

Antibodies, Monoclonal↗
PubMed1989

Quantitative histofluorescence of hippocampal mossy fiber zinc.

N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TS-Q) is a fluorescent probe for histochemically detecting a chelatable pool of zinc in the hippocampal formation and other brain regions. Utilization of zinc:TS-Q histofluorescence as a quantitative tool required further characterization of zinc:TS-Q fluorescence and the development of appropriate zinc standards. Studies of zinc:TS-Q fluorescence in aqueous solutions were conducted to optimize the reaction conditions for TS-Q chelation with zinc. Possible interference of zinc:TS-Q fluorescence by chelation of other biologically significant cations with zinc:TS-Q was shown to be negligible. Zinc standards were developed using a water soluble plastic embedding medium. Varying the concentration of zinc in the plastic sections yielded a standard curve exhibiting a log-linear relationship between 0.15 and 11 fg zinc/100 microns 2. Zinc:TS-Q histofluorescence was characterized in horizontal sections of rat ventral hippocampal formation using a computerized image analysis system. Section thickness and TS-Q concentration were optimized. The concentration of zinc varied between 1.5 and 17 fg/100 microns 2 in different regions of the mossy fiber projection of the ventral hippocampal formation. The development of this quantitative histofluorescence technique should facilitate our understanding of the functional role of zinc in the mossy fiber projection and can be utilized to investigate changes in mossy fiber zinc in a variety of pathological states.

Aminoquinolines↗
PubMedDate not supplied

Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue.

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.

Adrenocorticotropic Hormone↗
PubMed1992

Postnatal development of GABAergic neurons in the gerbil cochlear nucleus: pre-embedding and post-embedding immunocytochemical staining.

The purpose of this investigation was to compare the results obtained when plastic-embedded sections and vibratome-sliced sections were used to localize gamma-aminobutyric acid immunoreactivity (GABA-IR) in the gerbil cochlear nucleus (CN) during postnatal development. GABA-IR was mainly located in the perikarya of the neurons of the gerbil CN. At two days old, GABA-IR was found in plastic sections. No discernible GABA-IR was found in the vibratome slices at three to four days. At eight days, anti-GABA labeled cells were randomly located in the superficial and deep layer of the dorsal cochlear nucleus (DCN) in the vibratome sections, whereas they were mainly accumulated in the vicinity of the granule layer between the junction of the DCN and posteroventral cochlear nucleus (PVCN) in the plastic sections. At 14 days, anti-GABA labeled cells were reduced in number in the superficial third of the DCN in the vibratome slices and reduced in number in the junction between the DCN and PVCN in the plastic sections. At that time, a striking change was the formation of lamellation of the anti-GABA labeled cells mainly located in the middle third (fusiform layer) of the DCN in the plastic sections. A similar pattern of lamellation was found in the vibratome slices at 17-18 days. At three, four, six and eight weeks, the anti-GABA labeled cells were mainly located in the ventral part of the DCN close to the choroidal plexuses and the ventral part of the PVCN in the plastic sections. At six to 15 months old, distinct anti-GABA labeled cells were located in the fusiform layer of the DCN and scattered in the PVCN in the plastic sections. Another striking change at this age was that numerous vacuoles with a mesh-like network were present in the PVCN.

Animals↗
PubMed1975

[Rapid method of obtaining ultrathin sections in monolayer embedding].

It is proposed to employ a simple optical method for a correct relative adjustment of the knife and the roof of the piramid for obtaining ultrathin sections of monolayer embedding. For the material embedding, plastic cuvets and a lavsan film are employed, this technique facilitating the process of embedding.

Cytological Techniques↗
PubMed1980

Attempts to quantitate immunocytochemistry at the electron microscope level.

The ability to localize intracellular macromolecules in situ by high resolution techniques has been made possible by the development of antibody labelling of thin sections obtained either from tissues embedded in an hydrophilic matrix, or by ultracryotomy or from conventional plastic embedded tissue. When particle-tagged immunological reagents are used to visualize intracellular antigens, quantitative information can be obtained by combining particle counts with morphometric estimations of compartment volume. Various detection systems have been used successfully for quantitation, which include ferritin-conjugated antibodies, biotin-avidin-ferritin complexes and, more recently, gold-protein A conjugates. Examples of the use of these techniques the localization of secretory proteins in pancreatic exocrine cells, opsin and a large membrane protein in photoreceptor cells of frog retina, and contractile proteins in skeletal muscle are given. Quantitative data obtained by morphometric analysis, both in bovine and rat pancreatic exocrine cells, are compared with values assessed by biochemical methods.

Amylases↗
PubMed1981

"Pop-off" technic. The ultrastructure of paraffin-embedded sections.

Stained paraffin embedded sections sometimes contain a precise area that warrants further investigation at an ultrastructural level. A technic is described whereby an area in question may be reembedded into plastic and sectioned for examination under the electron microscope. The decoverslipped paraffin section is "popped-off" into an inverted BEEM capsule. Suspected viral inclusions in sections may be identified or ruled out with this technic. Special stains, especially heavy metal applications, not able to be utilized in conventional plastic embedding may be reembedded into plastic. The metallic granules may be visualized ultrastructurally without further grid staining. This method is especially helpful for cell smears and cell monolayers since they may be the only material available for study. Although ultrastructural detail is often poor, one does have the ability to study the identical section under both the light and the electron microscopes.

Glomerulonephritis↗
PubMed1990

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.

Animals↗
PubMed1989

Immunogold labeling method for Mycobacterium leprae-specific phenolic glycolipid in glutaraldehyde-osmium-fixed and Araldite-embedded leprosy lesions.

Phenolic glycolipid (PGL)-I, a Mycobacterium leprae-specific antigen currently used for serodiagnosis of preclinical leprosy, has thus far not been localized subcellularly in leprosy bacilli and their host cells. In this study, we developed an immunogold-labeling technique for qualitative identification of PGL-I sites in glutaraldehyde-osmium-fixed and Araldite-embedded M. leprae and host macrophages in human skin biopsies. Such "hard-fixed," plastic-embedded skin and nerve biopsies from patients with varying cell-mediated immunity to leprosy are amply available worldwide. Our method involves etching of plastic sections with H2O2, incubation with swine serum to eliminate nonspecific labeling, and long (22 hr) incubation at room temperature with monoclonal antibodies to PGL-I. Gold labeling was seen predominantly on cell walls of M. leprae, in vacuolar spaces of bacillated phagolysosomes, and occasionally on the cytoplasm and cell membrane of M. leprae. Host macrophage cytoplasm was labeled very infrequently. This technique allows studies on possibly persisting antigenic PGL-I in multibacillary leprosy patients during or after multidrug therapy. The method may also prove useful for subcellular localization of specific bacterial lipids in other mycobacterial diseases, including tuberculosis.

Antigens, Bacterial↗
PubMed1991

Myofiber orientation in the weanling mouse heart.

This study provides a quantitative description at the cellular level of myofiber orientation throughout the ventricles of the mouse heart. We employed computer-based methods of three-dimensional reconstruction from 3 microns plastic-embedded serial sections. Registration marks were introduced by drilling minute holes into each plastic block. Subfields of selected sections were photographed at 20 x magnification, using a computer-controlled microscope. The 35-mm film frames were projected onto a digitizer tablet and the epi- and endocardial boundaries were digitized manually. The "heads" and "tails" of linear segments of a representative myofiber sample present in each projected image were digitized in point mode. The many x-, y-, z-coordinate tables generated by digitization were reassembled automatically, giving a numerical description of the myofiber pattern. This pattern was studied interactively on a high-performance graphics workstation. We find that the heart wall is, to a first approximation, a "sandwich," in which the myofibers in the middle layer run mainly circumferentially, whereas those in the inner and outer layers run parallel or oblique to the apical-basal axis, a variant of the classical model of the myofiber pattern. We observed a "sleeve" in the interventricular septum, formed by longitudinal and oblique myofibers, a feature which apparently has not been described previously. Myofibers not running parallel to the transverse or longitudinal planes were not resolved in this study. We conclude that three-dimensional reconstruction of the cardiac myofiber pattern at the light-microscopic level, while laborious, is technically feasible and scientifically worthwhile.

Animals↗
PubMed1984

Quantitative evaluation of guinea pig spermatogenesis: Epon versus paraffin embedding.

A quantitative evaluation of spermatogenesis in the guinea pig was made at the light microscope level following improved methods of fixation and plastic embedding. The pattern of germ cell differentiation was studied by counting the average number of germ cell nuclei per Sertoli cell nucleus during the seminiferous epithelial cycle. Appreciable cell loss occurred during spermatogenesis. As a result, only 20.8% of the theoretically possible number of spermatozoa were produced from each spermatogonium. A slightly better yield of germ cells was obtained from Epon sections in comparison to paraffin sections. Epon sections can be effectively used for quantitative analyses of various cell populations in small biopsy specimens of testis tissue.

Animals↗
PubMed1986

Stabilization of fluorochromes after immunofluorescent staining of tissue and embedding in Epon.

Tissue blocks or sections immunofluorescent stained before embedding, i.g., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade less during illumination. This is an advantage of the pre-embedding, immunofluorescent staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.

Animals↗
PubMed1992

A double-embedding technique for thin tissue membranes.

A new double-embedding technique for thin tissue membranes is presented. This technique is useful for thin membranes such as mesenteric membranes from rodents, which usually measure only 10 microns in thickness. Several membranes are fixed and mounted on four needles located at the bottom of a plastic box. The box is filled with agarose at 50 C and then allowed to solidify. The agarose block is then removed, dehydrated in alcohol, cleared with HistoPetrol (isoparaffin hydrocarbons), permeated with paraffin and sectioned. The morphology is comparable to that obtained with methacrylate plastic embedding but is less time-consuming, less hazardous since no plastic hardener and activator are used and makes immunohistochemical studies easier.

Animals↗