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In Situ PCR Detection of HIV Expression in the Human Placenta

Control of vertical transmission of HIV-1 has progressed remarkably despite a lack of understanding of precise mechanisms by which infection may occur (1). The latter situation partially resulted from inadequate tools for in vitro analysis. However, in utero data suggest that placent ...

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Mesoderm Induction in Xenopus: Oocyte Expression System and Animal Cap Assay

Vertebrate mesoderm forms a diversity of tissues, including notochord, somitic muscle, pronephros, mesenchyme, and blood. In the amphibian Xenopus laevis, mesoderm arises in the equatorial region (marginal zone) of the blastula embryo in response to inducing signals produced by veg ...

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Improved Techniques for Avian Embryo Culture, Somite Cell Culture, and Microsurgery

For more than a century, avian embryos have been used in the study of development. Yet, as research techniques improve, there is increased need for animal models that exhibit excellent development in vitro and are easily accessible for observations, manipulations, and cell culturing. Here ...

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Amphibian Organizer Activity

The amphibian organizer corresponding to the upper dorsal blastopore lip of the early gastrula, is one of the classical systems in which embryonic patterning has been studied (1). Its two major inducing activities are neural induction and dorsalization of ventral mesoderm differenti ...

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Neural Crest Cell Outgrowth Cultures and the Analysis of Cell Migration

The neural crest gives rise to many differentiated cell types including craniofacial connective tissue, peripheral neurons, Schwann cells, various endocrine organs, and pigment cells and it has been extensively studied as a model of cell commitment and lineage diversification (1). H ...

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The Application of X-Ray Absorption Spectroscopy to Characterize Metal Centers in Proteins

The availability of synchrotron radiation has transformed the status of X-ray absorption spectroscopy (XAS) from a topic of relatively minor interest to one of major scientific importance and activity. The availability of a continuum of electromagnetic radiation with a high intens ...

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Double Indirect-Immunofluorescent Labeling of Cultured Cells

Immunofluorescence is a powerful technique for identifying and localizing intra- and extra-cellular components both in histological sections and in cultured cells of plant or animal origin. Briefly, an antibody, raised against a specific component, is used as a label to map the distribu ...

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Boyden Chamber Assay

The Boyden chamber assay, originally introduced by Boyden for the analysis of leukocyte chemotaxis, is based on a chamber of two medium-filled compartments separated by a microporous membrane. In general, cells are placed in the upper compartment and are allowed to migrate through the pores ...

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Isolation and Differentiation of Medaka Embryonic Stem Cells

Medaka is a small laboratory fish that daily produces eggs easily controllable by light cycles. This fish represents a unique lower vertebrate compared to mammals, in which embryonic stem (ES) cell lines can be derived from midblastula embryos (MBEs). Like mouse ES cells, medaka ES cells most res ...

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Use of Murine Embryonic Stem Cells in Embryotoxicity Assays: The Embryonic Stem Cell Test

The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their terat ...

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Preparation and Characterization of Glycolipid-Bearing Multilamellar and Unilamellar Liposomes

The carbohydrate residues of glycosphingolipids were implicated in many biologic processes such as cell-to-cell interactions; and as receptors for some viruses, bacterial and plant toxins, hormones, and so forth, and invariably for all the lectins (1). However, their receptor functi ...

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Purification and Characterization of Viral dsRNA Genome Profiles by Crosshybridization

Double-stranded (ds) RNA present as genetic elements are widely distributed in nature and include genomes of viruses that can infect both eukaryotic and prokaryotic cells. These include members of the family Reoviridae, Birnaviridae, Partitiviridae, Hypoviridae, Totiviridae, ...

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Primary Mouse Keratinocyte Culture

Mouse epidermal keratinocytes have traditionally been difficult to grow in vitro. In this chapter, we present a method for isolating epidermal keratinocytes from a single, newborn mouse pup for long-term culture. The protocols we describe will be especially useful for the isolation and a ...

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In Situ Hybridization with Radiolabeled cRNA Probes, Using Tissue Sections and Smears

Immunocytochemistry, together with biochemical procedures, has ensured rapid and accurate identification of tissue components. This has led to a better appreciation of cellular events, particularly storage and secretion of products. However, such techniques have certain dr ...

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Gene Mapping to Chromosomes by Hybridization in Situ

Since the technique of hybridizing labeled nucleic acid sequences to cytological preparations (hybridization in situ) was first described in 1969 by Pardue and Gall (1), it has undergone considerable refinement. In those early days, the technique was only capable of detecting and locat ...

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Immunogold Labeling for Electron Microscopy

Colloidal gold immunocytochemistry was first introduced by Faulk and Taylor (1), and has rapidly become a major technique in electron microscopy, covering many aspects of biological research. The rapid expansion of this technique in electron microscopy is first the result of its simpli ...

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Human Chromosome Analysis and Sorting

Flow cytometry has provided the cytogeneticist with a fast and accurate method of measuring the quantity of DNA in each human chromosome (1). Almost all the chromosomes in the human complement can now be resolved and abnormal chromosomes and aneuploidies (13,21, and X) recognized. A flow karyot ...

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Aging of Cultured Human Skin Fibroblasts

There is substantial evidence that aging is related to the finite ability of somatic cells to divide and repair damaged tissue. Since the seminal observation of Hayflick and Moor head (1) that cultured human fibroblasts have a finite replicative lifespan, a great deal of basic biological rese ...

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Transfection and Transformation of Human Thyroid Epithelial Cells

High efficiency gene transfer into mammalian fibroblasts is a routine procedure that can be performed by a variety of methods (1), but transfection of epithelial cells has been more difficult to achieve. Each of the standard techniques has associated problems; special equipment is requir ...

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Fluorescence-Activated Cell Sorting According to Receptor Density: Application for Isolating Transfected Cell Lines

Isolation of cells expressing transfected cDNAcan be extremely difficult and tedious if the efficiency of expression is low (less than 1–5%). If the receptor being selected for is a cell surface or integral membrane protein at least partly exposed to the exterior, then it is possible to use the extr ...

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