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PubMed1993

A method for producing unbiased histograms of neuronal profile sizes.

An important goal in neuroscience is to produce frequency distribution curves or histograms that relate numbers of cells to their sizes. Unfortunately such histograms, which are extremely common, are biased. Some of the reasons are inadequate attention to sampling paradigms, the lack of assurance that the section through the center of the cell is measured, and that large cells will have more profiles than small cells. The first goal of this paper is to illustrate these biases for dorsal root ganglion cells by showing significant statistical differences between histograms prepared the classic way and histograms produced by unbiased methods. The differences are particularly dramatic for plastic embedded material. The second goal of the paper is to describe an unbiased method for obtaining these histograms. The procedure is 2-fold. Cells are chosen in an unbiased way (unbiased in this sense means that every cell has an equal chance of being chosen), and then the largest profile of the cell is measured. We further suggest that these histograms be accompanied by analyses of cell volume, since volumes of cells rather than diameters or areas of cell profiles, will be the future measure of choice when considering the sizes of cells (or any other particles of biologic interest).

Animals↗
PubMed1985

A simple re-embedding method for the preparation of testicular cell cultures for light and electron microscopy.

A simple and reproducible method is described for the preparation of cell cultures for light and transmission electron microscopy. Mixed testicular cells are grown on collagen layered, PTFE-coated coverslips. Thin sheets of plastic-embedded cells are packed together and re-embedded permitting the examination of sections cut perpendicular to the plane of the culture and, thus, the intercellular relationships at different levels throughout the culture.

Animals↗
PubMed1989

High frequency of in situ hybridization on thin plastic sections of human placenta with a cDNA probe for beta hCG.

We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.

Autoradiography↗
PubMed1986

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques.

Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling. Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate. The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity. The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.

Animals↗
PubMed1985

Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti-opsin antibodies.

We have used a cytochemical technique for labeling freeze-fractured tissues (Pinto da Silva, P., C. Parkison, and N. Dwyer (1981) Proc. Natl. Acad. Sci. U.S.A. 78: 343-347) to examine the distribution of immunoreactive opsin in rod photoreceptor membranes. Aldehyde-fixed retinas of African clawed frogs (Xenopus laevis) embedded in a cross-linked protein matrix were frozen and fractured at -196 degrees C, then thawed and labeled with biotinylated sheep anti-cow opsin IgG followed by avidin-ferritin. In thin sections of plastic-embedded retinas, rod outer segment (ROS) disc membranes exposed by fracturing bound specific antibody intensely and relatively uniformly. However, they differed from membranes of the inner segment as well as those of erythrocytes in that protoplasmic face leaflets did not assume an interrupted bilayer appearance and disc exoplasmic face leaflets were apparently lost during thawing. The disposition of opsin immunoreactivity in the cell membrane was highly asymmetric. Although ROS plasma membranes from which discs are elaborated labeled heavily with anti-opsin after cleavage, fractures passing along inner segment plasma membranes bound very little antibody. In cross-fractures exposing inner segment cytoplasm, we found specific labeling of Golgi complex elements, as well as both perimitochondrial and periciliary vesicles. The latter are presumed to be the vehicle shuttling newly synthesized membrane to the ROS for disc assembly. These results suggest that opsin-containing membrane is sorted out within the cell, being transported from synthetic sites to the immediate periciliary zone where localized insertion into the cell membrane takes place. Furthermore, the close correspondence of the present immunocytochemical analysis with the distribution of opsin deduced from prior quantitative freeze-fracture analysis (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463) offers the possibility that fracture-label may be generally useful for study of patterned membrane topography in neuronal cells.

Animals↗
PubMed1984

Primary culture of chick embryo skeletal muscle on dextran microcarrier.

We report the conditions to obtain primary suspension cultures using embryonic skeletal muscle from 12-day chick breast muscle. Further, the conditions are described to obtain scanning electron micrographs of whole cells and transmission electron micrographs of sections of plastic-embedded cells on microcarriers. A positively charged hydrated dextran microcarrier, Cytodex I (Pharmacia), provided support for the cells; the myogenic stages of proliferation, myoblast alignment and fusion to form myotubes coincided temporally with replicate cultures grown on gelatin-coated plastic dishes. Microcarrier-grown cells, including non-muscle cells, had microvilli, lamellipodia, bleb, and other surface modifications but no ruffling membranes. Myoblasts and myotubes on beads had fewer microvilli compared to homologous cells grown in the static culture medium of plastic dishes. Myoblasts aligned laterally during fusion, starting at 48 h. Myotube cytodifferentiation proceeded to myofibril formation by day 4 of microcarrier culture. The sarcomeres of aligned myofibrils had normal banding with an hexagonal lattice of thick and thin myofilaments in the A-bands. Caveolae intracellulares and sarcoplasmic reticulum were evident. Scaling-up to larger volumes promises to provide a cost-effective way to obtain a large harvest of cultured skeletal muscle which may prove especially useful for studies of minor constituents.

Animals↗
PubMed2001

A new sample preparation method for biological soft X-ray microscopy: nitrogen-based contrast and radiation tolerance properties of glycol methacrylate-embedded and sectioned tissue.

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 10(8) Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.

Animals↗
PubMed1998

Fate of Schwann cells in CMT1A and HNPP: evidence for apoptosis.

The fate of Schwann cells in Charcot-Marie-Tooth (CMT) neuropathies was addressed in this study of nerve biopsies from patients with proven PMP22 duplications and deletions. In frozen sections, apoptotic nuclei were detected using the TUNEL method. In adjacent sections, anti-neurofilament 68kD antibody was used as an axonal marker, while the antibodies to NKH-1 and low-affinity nerve growth factor receptor P75NTR were used as Schwann cell markers. In addition, plastic sections were used to determine the densities of myelinated fibers and Schwann cell nuclei. In all biopsies from CMT1A, TUNEL-positive nuclei appeared in clusters. In adjacent sections, areas of TUNEL-positive nuclei matched with areas devoid of neurofilaments and NKH-1-positive Schwann cell silhouettes, suggesting that the apoptotic nuclei belonged to nonmyelinating Schwann cells. In addition, quantitative studies on plastic-embedded sections showed a significantly reduced number of total Schwann cells compared with controls, strongly favoring a loss of Schwann cell by apoptosis. In HNPP, the number of total Schwann cells was increased and a significant Schwann cell apoptosis was observed in only 2 patients. Examination of plastic sections and teased nerve preparations from these cases suggested that the Schwann cell apoptosis might be related to the regenerative state of the nerve resulting from the process of sprout pruning. No strict correlation between p75NTR expression and apoptosis was found. These studies indicate that factors regulating Schwann cell number in early postnatal development continue to be important for Schwann cell survival throughout life.

Adult↗
PubMedDate not supplied

Enzyme and immunohistochemistry of follicular hyperplasia in AIDS-related lymphadenopathy.

We used a panel of monoclonal and polyclonal antibodies to analyze frozen and paraffin-embedded lymph node biopsy specimens from 25 intravenous drug abusers (IVDA) with acquired immunodeficiency syndrome (AIDS)-related lymphadenopathy histologically characterized by follicular hyperplasia. Our aim was to obtain diagnostic clues to this commonly occurring pattern. Double-labelling immunohistological studies were also performed on selected frozen sections and 13 plastic-embedded specimens were tested by a number of enzyme reactions. Consistent features in IVDA included abnormally high numbers of intrafollicular T-cells, positive for acid phosphatase and beta-glucuronidase, most of which had Leu-2a-positive phenotype; a marked reduction or loss of mantle zone B-cells (positive for surface IgD-IgM and alkaline phosphatase); and disarray of the network of follicular dendritic reticulum cells (DRCs), as revealed with DRC-1 and anti-S-100 protein antibodies or with reaction for 5'-nucleotidase. When present, distinctive intrafollicular clusters of Leu-2a-positive T-cells and mantle zone B-cells were nearly always associated with areas lacking DRCs in some patients. The intrafollicular hypervascularity invariably found in IVDA proved to be of a true capillary nature, as demonstrated by alkaline phosphatase, 5'-nucleotidase, and ATPase reactions. In control tissues, all showing absence of Leu-2a-positive intrafollicular T-cells, most of the above individual changes could be detected, although they were occasional, mild, and never associated within the same follicle. By contrast, combined immunohistological and enzyme histochemical findings in IVDA indicated that in most follicles such changes were marked and very often associated within the same follicle in each case.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Related Complex↗
PubMedDate not supplied

The low temperature vacuum embedding technique for X-ray microanalysis of the developing inner ear.

The method of low-temperature embedding in vacuo using methacrylate resins, was tested for X-ray microanalysis of the embryonic inner ear of the CBA/CBA mouse. Fetal inner ears were examined on gestational days 16 and 18. The technique was evaluated in comparison with earlier used preparation techniques for X-ray microanalysis. With plastic embedding, an improvement of the morphological resolution was achieved, which allows reliable structural identification on the subcellular level. Furthermore, the possibility of orientating of the specimen prior to sectioning provides a better controlled and less time-consuming sectioning.

Animals↗
PubMed1997

Apoptosis in male germinal line cells of rat in vivo: caused by phosphamidon.

When Wistar strain male albino rats were fed on drinking water containing phosphamidon at 35 ppm concentration for 30 days, the spermatogenic compartment of the testis underwent histopathological changes. Differentiating spermatids and fully differentiated spermatozoa were rare or absent. Uni- and multinucleate giant cells appeared in the epithelium as well as the lumen. Semithin sections of resin-embedded tissues revealed that such cells had a typical apoptotic morphology. These cells were traceable to the caput and cauda epididymidal duct, and in the latter they appeared to fragment into apoptotic bodies. The male reproductive toxicity of phosphamidon leading to death and removal of the spermatogenic line cells through apoptosis is described. It is suggested that plastic embedding and semithin sectioning are appropriate methodologies to identify apoptotic cells in histological preparations viewed by light microscopy.

Animals↗
PubMed1995

Histochemistry and immunohistochemistry on bone marrow biopsies. A rapid procedure for methyl methacrylate embedding.

Starting from previous methodical approaches a procedure for low temperature methyl methacrylate (MMA) embedding of bone marrow biopsies is introduced, which allows routine application of enzyme and immunohistochemistry without loss of morphological quality by retaining fixation in Schaffer's solution. Survival of enzyme activity and antigen determinants is achieved by washing the fixed specimens in 70% methanol and dehydration in acetone in ascending concentrations at 4 degrees C. Modifications of the plastic embedding technique used in this study simplify and shorten the procedure, so that embedding according to this routine method is complete after two days of preparation. Additionally, a rapid embedding variant is introduced, which enables tissue preparation within one day if necessary. Results are demonstrated using markers for myeloid, lymphoid and epithelial cells as well as immunoglobulins and the proliferation associated antigen Ki-67. The investigation of a panel of monoclonal and polyspecific antibodies in 31 cases shows the eventually reduced immunoreactivity of a few markers after prolonged fixation. As a consequence it seems essential to ensure short fixation periods, especially when the specimens are sent by mail.

Biopsy↗
PubMed1985

Germ cell quantitation in human testicular biopsy.

Quantitative analysis of human seminiferous epithelium was carried out using an improved method of glutaraldehyde and osmium fixation with plastic embedding. Part of each biopsy specimen was fixed in Bouin's fixative and embedded in paraffin for comparison. Epon embedded tissue had very little artifactual damage compared with paraffin embedded tissue sections. The germ cell to Sertoli cell ratios were determined by counting the various germ cells per "unit" tubular area. Data obtained by this method reflect a remarkable stability of Sertoli cell number and germ cell-Sertoli cell ratios both between biopsies from different individuals and between biopsies from right and left testes from the same individual. Agreement between the present results and those of earlier studies based on paraffin embedded testicular specimens supports the validity of this method of germ cell quantitation of human testicular biopsy samples.

Adolescent↗
PubMed1976

Improved microradiographic contrast for bone stain-historadiography.

Microradiographs of 5-micron sections of methyl methacrylate embedded undemineralized bone show poor resolution, but prestaining with silver nitrate increases the radioopacity of the calcified tissues to soft x-rays without masking regional differences in microscopic mineralization. Identical results are achieved using aqueous (pH 5.8 and 7.5) or ammoniacal solutions (pH 7.9). Atomic absorption spectrometry detected no loss of calcium from the sections during staining. Osteoid in plastic-embedded bone is not rendered radiopaque by this technique even after prolonged staining times (5 min-2 hr).

Autoradiography↗
PubMed2006

Operant matching is a generic outcome of synaptic plasticity based on the covariance between reward and neural activity.

The probability of choosing an alternative in a long sequence of repeated choices is proportional to the total reward derived from that alternative, a phenomenon known as Herrnstein's matching law. This behavior is remarkably conserved across species and experimental conditions, but its underlying neural mechanisms still are unknown. Here, we propose a neural explanation of this empirical law of behavior. We hypothesize that there are forms of synaptic plasticity driven by the covariance between reward and neural activity and prove mathematically that matching is a generic outcome of such plasticity. Two hypothetical types of synaptic plasticity, embedded in decision-making neural network models, are shown to yield matching behavior in numerical simulations, in accord with our general theorem. We show how this class of models can be tested experimentally by making reward not only contingent on the choices of the subject but also directly contingent on fluctuations in neural activity. Maximization is shown to be a generic outcome of synaptic plasticity driven by the sum of the covariances between reward and all past neural activities.

Animals↗
PubMed1989

Specific demonstration of ribonucleic acid by chemical bromination and immunohistochemistry.

In this report we describe a specific staining procedure for detection of ribonucleic acid (RNA), based on bromination of uracil and subsequent immunohistochemical visualization of 5-bromouracil in RNA. This method is applicable for both cryostat and glycol methacrylate (GMA)-embedded sections. Cryostat sections must be fixed in formaldehyde, whereas tissue pieces to be embedded in GMA are fixed in cold acetone. Before bromination, sections must be treated with trypsin. Bromination was performed in a solution of bromine in potassium bromide. After bromination, excess bromine was removed with sodium bisulfite. The monoclonal antibody MoBu-1 specifically bound to brominated RNA. Ribonuclease digestion, in contrast to deoxyribonuclease digestion, abolished staining. This method makes possible precise localization of RNA, especially well demonstrated in plastic-embedded sections.

Animals↗
PubMed2000

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes.

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.

Archaeal Proteins↗