bluuebunny/crossref_metadata_2025_split huggingface dataset:
Applications of immunogold‐silver enhancement: Testing of monoclonal antibodies and detection of human cytomegalovirus in histologic specimens
Authors: Dankner Wayne M. , Spector Stephen A. 
Published: February 2005

AbstractHuman cytomegalovirus (HCM‐V) is an important pathogen in neonates, transplant patients, and individuals with acquired immunodeficiency syndrome (AIDS). Reliable techniques for the detection of this virus in clinical specimens would aid in improving methods for diagnosis and increasing our understanding of viral pathogenesis. We evaluated the utility of immunogold‐silver enhancement to determine the specificity of murine monoclonal antibodies directed against HCMV proteins and applied these antibodies to the detection of HCMV in histologic material. Nine antibodies were tested in a tissue‐culture system to determine the location of staining. All were found to be active in frozen tissue, but only two of these antibodies were reactive in formalin‐fixed tissue. We also evaluated a novel tissue fixative technique (AMeX; Fixation and dehydration with acetone) that has been used to maintain antigenicity of T‐lymphocyte cell markers. All antibodies remained reactive against their respective HCMV proteins in tissue fixed by this technique. However, dehydration of tissue may limit the usefulness of AMeX fixation. Immunogold‐silver enhancement is a useful and reliable histochemical technique for detection of HCMV antigens in pathologic specimens.
Use of immunogold preembedding technique to detect hepatitis A viral antigen in infected cells
Authors: Cromeans Theresa , Humphrey Charles , Sobsey Mark , Fields Howard 
Published: February 2005

AbstractLocalization of virus and viral antigen in cell cultures infected with a rapidly replicating isolate of strain HM‐175 of hepatitis A virus (HAV; pHM‐175) was accomplished by using immunogold probes. Cells infected under one‐step growth curve conditions were prefixed with 2% paraformaldehyde and 0.1–0.001% saponin at appropriate times postinfection for detection of maximum virus and viral antigen. An indirect labeling technique was employed using monoclonal antibody to HAV followed by 5 nm gold‐antimouse IgG conjugate. Cells were then fixed by standard electron microscopy techniques and thin sectioned. This prefixation technique allowed penetration of the immunogold probes and moderate preservation of ultrastructure. Within infected cell cytoplasm, numerous antigenic sites were labeled with six to 200 gold particles. Two types of cells were infected with HAV and somewhat different results were obtained with the two cell types. In BS‐C‐1 cells, where a cytopathic effect (CPE) was not observed, myelin figures were immunogold labeled or frequently were located near immunogold‐labeled sites. Vesicles containing viruslike particles (14–22 nm) were also observed. A significant observation in infected FRhK‐4 cells was the presence of multivesicular bodies labeled with immunogold. Microfilaments were commonly seen near the multivesicular bodies. Our results demonstrate that the choice of prefixation method for immunogold labeling should be empirically determined for the cell type and condition.
Immunogold‐silver labeling in histology
Authors: In't Veld Peter A. 
Published: February 2005

AbstractImmunogold‐silver staining is a relatively new technique in light‐microscopic immunocytochemistry. It is based on silver enhancement of 1 to 10 nm gold probes immunologically bound to antigenic determinants in tissue sections. The detection efficiency of this third‐generation detection technique is compared to that of the peroxidase‐based ABC technique in routinely processed, paraffin‐embedded, human biopsy material.
Immunogold‐labeled microtubule crossbridges in platinum‐carbon replicas of the marginal band of erythrocyte cytoskeletons
Authors: Centonze Victoria E. , Ruben George C. , Sloboda Roger D. 
Published: February 2005

AbstractCytoskeletons of erythrocytes from the toad Bufo marinus are composed of a surface‐associated cytoskeleton that encapsulates the annular bundle of microtubules known as the marginal band (MB) and the centrally located nucleus. As seen by phase‐contrast microscopy, the microtubules (MTs) of the MB remain tightly bun‐led after cell lysis without the need for added stabilizing factors. The integrity of this structure suggested that in addition to MTs other components were present in the MB and were responsible for its stability. Thin (< 18 nm) platinum‐carbon (Pt‐C) replicas of freeze‐dried cytoskeletons prepared by using a modified Balzers 300 system provided a novel method of sample reparation for a high‐resolution study of the ultrastructure of the MB. Electron micrographs of replicas revealed that, the MTs of the MB displayed numerous filamentous projections which, hen viewed in stereo, appear as side‐arm connections between adjacent MTs. Immunofluorescence data show that monospecific antibodies to tubulin and to MT‐associated protein 2 (MAP2) from brain each detect cross‐reactive material in the MB. The combination of immunogold cytochemistry with Pt‐C replication provided the increased resolution required to identify the individual structures recognized by antibodies to tubulin and MAP2. As expected, antitubulin labeled the MTs of the MB. However, anti‐MAP2 antibodies were localized specifically to the cross‐ridging filaments between adjacent MTs. Thus, a IAP2‐like protein was identified in situ as the crossbridging filament that bundles MTs to form a table MB.
On‐grid immunogold labeling of glial intermediate filaments in epoxy‐embedded tissue
Authors: Johnson Anne B. , Bettica Annemarie 
Published: February 2005

AbstractOn‐grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold‐labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy‐embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde‐fixed, epoxy‐embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on‐grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween‐20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy‐embedded material to immunolabel thin structures like intermediate filaments.
Light and electron microscopic observations on the cervical epithelium of the rabbit: II
Authors: Odor D. Louise , Horacek Mark J. , Blandau Richard J. 
Published: February 2005

AbstractThe endocervical epithelium of long‐term ovariectomized rabbits treated for 1–10 days with 5 μg of estradiol benzoate every 12 hr has been studied by light and electron microcopy. In addition, morphometric data on ciliated and nonciliated cells of rabbits treated for 2, 6, and 10 days are compared to those on untreated ovariectomized, estrous, and ovulatory rabbits.The percentage of ciliated cells increases after ovariectomy to 76.3% and that of secretory cells decreases to 23.7% as compared to estrous controls. Treatment of ovariectomized rabbits with estradiol results in a gradual increase in ciliated and secretory cell area, height, and nuclear area. After 10 days of treatment, cell areas are significantly larger than those in the ovulatory or estrous controls; cell height and nuclear areas have returned to preovariectomized levels; and the percentages of ciliated and secretory cells have reached those of estrous levels. Estradiol stimulates mitotic division of secretory cells but affects ciliogenesis minimally.In ciliated cells, estradiol treatment results in a modest increase in polysomes and granular endo‐plasmic reticulum and in striking increases in the size of the Golgi complex and in the number of lipofuscin bodies as compared to those in the ovariectomized controls.In secretory cells, estradiol treatment brings about an increase in the numbers of polysomes, Golgi complexes, and cisternae of the granular en‐doplasmic reticulum, in the sizes of the nucleoli, and in the amount of euchromatin. Secretory granules appear in some cells after 2 days of estradiol stimulation and increase in number through 10 days of treatment. Perinuclear granules are more pleomorphic and heterogeneous in structure and more numerous in the 6‐ to 10‐day‐estradiol‐treated than in ovulatory animals, and they may function as lysosomes degrading excess secretory product. Deep apical concavities of the secretory cells occur most often after 2 and 6 days of treatment.
Mediobasal prosencephalic defects, including holoprosencephaly and cyclopia, in relation to the development of the human forebrain
Authors: Müller Fabiola , O'Rahilly Ronan 
Published: February 2005

AbstractFour very early synophthalmic embryos were studied in serial sections and reconstructed graphically by the point‐plotting method. Three belonged to stage 16 (5 weeks) and one to stages 19/20 (7 weeks). Recently completed accounts and reconstructions of the normal brains of staged human embryos served as controls for comparison with the abnormal examples. The embryos shared in common: holoprosenceph‐aly, arhinencephaly sensu stricto (absence of olfactory nerve fibers, bulbs, and tracts), presence of a proboscis, synophthalmia with two lens vesicles, a retarded telencephalic wall, absence of the mediobasal part of the telencephalon (the future septal area and the commissural plate: future anterior commissure and corpus callosum), irregularity of the diencephalon, mensural changes in the brain, absence of the rostral part of the noto‐chord and consequent cranial defects, and small ganglia of the cranial nerves. Where it could be determined (at least in the three less advanced specimens), the adenohypophysial primordium was either small and isolated or was absent; a ten‐torial condensation appeared to be missing; and disturbances of the primordia of the orbital muscles and their innervation were noted. The corpus striatum is single and corresponds to only the di‐encephalic part (medial eminence) of normal embryos. Interference with induction by the pre‐chordal plate at or before stage 8 (18 days) would be expected to affect the future mediobasal part of the neural plate (median prosencephalic dysgen‐esis) and the future optic primordium (cyclopia sensu stricto). Insufficient formation of material from the prechordal plate would account for disorders of the orbital musculature and, possibly, for inadequacy of the tentorium cerebelli. Disturbance a couple of days later (stage 9) would result in synophthalmia. Cyclopia and synophthalmia entail arhinencephaly and holoprosencephaly, both of which may arise independently. Defective distribution of the cephalic mesenchyme points to a derangement of the mesencephalic neural crest (stages 10 and 11), causing such features as an incomplete chondrocranium and reduction in size of the ganglia of the cranial nerves. Failure of bilateral division of the telencephalon would occur at or before 4 weeks (stages 13 and 14). It is concluded that all the above conditions arise during the first 4 postovulatory weeks.
Individual rabbit cardiac myocytes have different thresholds for alpha myosin heavy chain regulation by thyroid hormone
Authors: Lin Zhaoying W. , Wenderoth Mary P. , Eisenberg Brenda R. 
Published: February 2005

AbstractMyocytes in adult rabbit ventricle express an α and a β form of myosin heavy chain (MHC). The α‐MHC distribution detected with indirect immunofluorescence has been found in different proportions in adjacent myocytes producing a mosaic staining pattern. The basis for cell‐specific expression of the α‐MHC isoform is not known. Since thyroid hormone is a major regulator of myosin gene expression, we varied the plasma thyroid level and followed the α‐MHC content within a population of myocytes. Ventricular myocytes were induced to become 100% β‐MHC by placing the rabbits on a 0.15% propylthiouracil diet for 70 days. L‐triiodothyronine (LT3) over a dose range of 1 to 10 μg/kg/day was delivered by an osmotic minipump for 5 days, with actual serum levels confirmed by LT3 radioimmunoassay to be in the range of from 115 to 1,230 ng/dl. The amount of α‐MHC that returned was estimated in randomly selected cells by measuring the relative intensity of the fluorescence‐tagged secondary antibody. The normal mosaic pattern of α‐MHC expression in the left ventricle returned with an LT3 dose of 2–5 μg/kg/day. The first myocytes to express α‐MHC were in the subepicardium and did so at a LT3 serum level of 115 of ng/dl. All myocytes of the ventricular wall expressed α‐MHC at serum levels above 1,230 ng/dl. These data are interpreted to show that the variation of myosin isoform content seen in the adult heart is indicative of heterogeneity of thyroid sensitivity, with the threshold for serum LT3 being between 115 and 370 ng/dl.
Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: I. Normal development
Authors: Brody Joel R. , Cunha Gerald R. 
Published: February 2005

AbstractMyometrial development from the prenatal to adult period was examined in rats and mice (1) by histologic and immunocytochemical methods with anti‐actin, ‐vimentin, and ‐laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells; and (2) by morphometric procedures to assess quantitatively the expression of cellular orientation in the emerging inner circular myometrial layer. Uterine mesenchymal cells initially were uniformly vimentin‐positive, undifferentiated, and randomly oriented during the late fetal period. By the early neonatal period, three mesenchymal layers became recognizable histologically, the middle one of which (prospective circular myometrium) developed distinct circular orientation and differentiated into a layer composed of actin‐positive smooth muscle cells. The cells of the inner mesenchymal layer initially exhibited radial orientation. By 10 days postpartum, the outer longitudinal mesenchymal layer differentiated into bundles of smooth muscle cells representing the longitudinal myometrium. The inner mesenchymal layer remained vimentin‐positive and differentiated into the randomly ordered endometrial stroma. The cells of the middle and outer mesenchymal layers that were destined to form myometrium initially expressed vimentin throughout and then coexpressed vimentin and actin, but with time vimentin staining disappeared in the maturing smooth muscle cells as they expressed actin.
Trophoblast differentiation during the transition from trophoblastic plate to lacunar stage of implantation in the rhesus monkey and human
Authors: Enders Allen C. 
Published: February 2005

AbstractThe transition from the tropho‐blastic plate stage to the early lacunar stage was examined in a series of implantation sites from the rhesus monkey, timed on the basis of the preovulatory estrogen peak, and prepared for transmission electron microscopy. This transition was compared with specimens from stage 5a, b, and c in the Carnegie collection of human embryos. The transition was marked by the differentiation of a new type of syncytial trophoblast—namely, a unilaminar microvillous polarized syncytium, which developed throughout the trophoblastic plate, forming characteristic intrasyncytial clefts. The rapid development of this type of syncytium created a nonclotting chamber for maternal blood wherever trophoblast intrusion into maternal vessels created confluence. Although the nature of the material in the Carnegie series precluded cytological characterization of the trophoblast, there is evidence that a similar transition occurs in human trophoblast and that in the human also the appearance of lacunae marks a change from an early invasive trophoblast to a situation in which growth is more significant.
Reconstitution of endoplasmic reticulum in rapidly dividing cells of early Xenopus embryos
Authors: Dominguez J. Manuel , Paiement Jacques 
Published: February 2005

AbstractThe cytology of early blastomeres of Xenopus laevis embryos was examined. Particular attention was given to the organization of the nuclear envelope of karyomeres (chromosome vesicles) and the endoplasmic reticulum (ER) at different stages in early cleavage cycles of frog development. Nuclear envelope formation was observed to occur rapidly around individual chromosomes during early anaphase, and karyomeres fused subsequently to yield the final nucleus during telophase. Endoplasmic reticulum in the perinuclear cytoplasm was observed to be vesicular during metaphase and cisternal in form during telophase. Following microinjection of rat liver rough microsomes into early blastomeres, heterologous ER components were identified by electron microscope immunocyto‐chemistry. The foreign ER was observed as large, reconstituted cisternae at stages in the cell cycle when the nuclear envelope was intact. Therefore, transplanted ER maintained the capacity to reconstitute in the cytoplasm of a rapidly dividing cell. In an attempt to better assess ER structure at the metaphase stage of the cell cycle, we next slowed down the division process by treating Xenopus embryos with anti‐microtubule agents. Treatment with critical concentrations of colchicine, nocodazole, or vinblastine led to cleavage arrest but not to inhibition of the nuclear cycle. Following such treatment, homologous ER was observed in a vesicular form at all stages of the nuclear cycle. Heterologous ER, however, identified by immunocyto‐chemistry in microinjected cells treated with nocodazole, displayed both vesicular and cisternal forms. We conclude that microinjected ER membranes exhibit cell‐cycle‐specific behavior, which is different from that of the host cell ER.
Endothelial attachment and plasmalemmal apposition in the transcellular movement of intravascular leukemic cells entering the myeloid parenchyma
Authors: De Bruyn Peter P. H. , Cho Yongock , Michelson Sonia 
Published: February 2005

AbstractThe plasmalemmal relationship between metastases‐forming leukemia cells and myeloid sinus endothelium during the transmural passage of the leukemia cells has been studied in rat bone marrow. After the myeloid vascular system was freed from normal circulating blood cells, the bone marrow was perfused with a suspension of leukemia cells derived from an ascites tumor. The bone marrow was then fixed by perfusion with double aldehyde with and without the addition of tannic acid.Leukemia cells were seen adhering to the adluminal aspect of the sinus endothelium and in all stages of endothelial penetration. The penetration of the sinus wall was independent of endothelial junctions; i.e., the transmural passage into the myeloid parenchyma was transcellular. At these sites, there were restricted areas of close plasmalemmal appositions of the two cell types where the intraplasmalemmal space was reduced to 2.3 nm. This space was interrupted by electron densities of 5 nm diameter and spaced 9 nm center to center. These close plasmalemmal appositions extended over distances ranging from 150 nm to 200 nm. It is suggested on the basis of the structural similarity that these heptalaminar complexes of close plasmalemmal apposition represent the structural equivalent of gap junctions and may be sites of intercellular communication requisite for transmural passage.When tannic acid was added to the fixative, there were extended areas of apparent fusion of the plasmalemmas of the two cell types, at the sites both of adhesion and of endothelial penetration. This fusion was limited to the outer leaflets of the two plasmalemmas, resulting in a single pentalaminar complex. These pentalaminar complexes extended over decidedly longer distances than the presumed gap junctions seen in the non‐tannic‐acid‐fixed material. The tannic acid material did not show the heptalaminar gap junction type of plasmalemmal apposition. It is believed likely that the tannic‐acid‐induced pentalaminar complexes may incorporate the smaller heptalaminar ones. The factors underlying the plasmalemmal configurational differences between the tannic acid and non‐tannic‐acid material remain undetermined.
Role of nerve and muscle factors in the development of rat muscle spindles
Authors: Kucera Jan , Walro Jon M. , Reichler Judith 
Published: February 2005

AbstractThe soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent‐muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (α) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (γ) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when γ axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not γ motor innervation. However, transient innervation of future bag2 fibers by α axons suggests that both sensory and α motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.