The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.

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 JoVE Immunology and Infection

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles


JoVE 4023 3/15/2012

Institute of Biochemical Engineering, Technische Universität Braunschweig

A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.

 JoVE Clinical and Translational Medicine

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation


JoVE 50247 2/26/2013

1Department of Cardiac Electrophysiology, Piedmont Heart Institute, 2Cardiac Rhythm, Medtronic Inc.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

 JoVE Neuroscience

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds


JoVE 2389 2/15/2011

1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

 JoVE Neuroscience

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons


JoVE 3200 9/15/2011

1Institute for Cellmorphology and molecular Neurobiology, Group for Cellbiology, Ruhr-University Bochum, 2Institute for Clinical Neurobiology, University of Wuerzburg

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

 JoVE Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering


JoVE 3366 11/25/2011

Biomedical Engineering Department, Worcester Polytechnic Institute

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

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 JoVE General

Double Whole Mount in situ Hybridization of Early Chick Embryos


JoVE 904 10/27/2008

1Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology - Texas A&M Health Science Center, 2Center for Environmental and Genetic Medicine, Texas A&M University (TAMU)

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

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 JoVE General

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber


JoVE 3415 2/13/2012

1Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School, 2Department of Physiology and Biophysics, Robert Wood Johnson Medical School, 3Muscle Biology Research Group-MUBIG Schools of Nursing & Medicine, University of Missouri-Kansas City

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

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 JoVE Clinical and Translational Medicine

Myo-mechanical Analysis of Isolated Skeletal Muscle


JoVE 2582 2/22/2011

1Cardiovascular Research Institute, University of California San Francisco, 2Department of Pediatrics, University of California San Francisco, 3Department of Biology, San Francisco State University, 4Department of Medicine, University of California San Francisco, 5Eli and Edythe Broad Center of Regeneration Medicine & Stem Cell Research, University of California San Francisco

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

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 JoVE Neuroscience

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI


JoVE 4055 7/12/2012

1Department of Radiology, Stanford University, 2Center for In Vivo Microscopy, Duke University Medical Center, 3Department of Biomedical Engineering, Duke University

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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 JoVE General

Assay for Neural Induction in the Chick Embryo


JoVE 1027 2/13/2009

Institute of Biosciences and Technology, Center for Environmental and Genetic Medicine, Texas A&M University (TAMU)

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

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 JoVE Neuroscience

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae


JoVE 3837 5/18/2012

1Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, 2Scholars Academy/MARC Scholar, University of Houston-Downtown, 3Genes and Development Graduate Program, University of Texas Graduate School of Biomedical Sciences, 4Neuroscience Graduate Program, University of Texas Graduate School of Biomedical Sciences

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

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 JoVE Clinical and Translational Medicine

In vitro Measurements of Tracheal Constriction Using Mice


JoVE 3703 6/25/2012

Department of Physiology, UT Health Science Center, San Antonio

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

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 JoVE General

A Chromatin Assay for Human Brain Tissue


JoVE 717 3/21/2008

Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

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 JoVE Applied Physics

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition


JoVE 50297 2/27/2013

1Department of Energy and NEMAS - Center for NanoEngineered Materials and Surfaces, Politecnico di Milano, 2Center for Nano Science and Technology, Instituto Italiano di Tecnologia

We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.

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 JoVE Neuroscience

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales


JoVE 2354 11/14/2010

1Department of Cell Biology and Neuroscience, Rutgers University, 2Graduate Program in Biomedical Engineering, Rutgers University

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

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 JoVE Clinical and Translational Medicine

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice


JoVE 3260 11/21/2011

1Division of Surgical Research, University Hospital Zurich, 2Institute of Laboratory Animal Science, University of Zurich

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

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 JoVE Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin


JoVE 2315 12/23/2010

Microbiology and Immunology, James Graham Brown Cancer Center, University of Louisville

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

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 JoVE Clinical and Translational Medicine

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography


JoVE 3119 9/22/2011

1Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, 2Department of Biology, North Carolina Central University, Durham, 3Department of Physiology & Pharmacology and Hypertension & Vascular Research Center, Wake Forest University School of Medicine

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.

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 JoVE General

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF


JoVE 1550 11/18/2009

1Research and Development, Stemgent, 2Product Marketing, Stemgent

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

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 JoVE Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons


JoVE 2794 7/13/2011

1Department of Physiology, Northwestern University Feinberg School of Medicine, 2Department of Psychiatry and Behavioral Sciences, Northwestern University Feinberg School of Medicine

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

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 JoVE Bioengineering

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography


JoVE 4390 1/15/2013

1Department of Biomedical Engineering, Northwestern University, 2Department of Physics, Harbin Institute of Technology, 3Department of Ophthalmology, University of Southern California, 4Department of Ophthalmology, Northwestern University

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

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 JoVE Clinical and Translational Medicine

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer


JoVE 50198 2/08/2013

1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

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 JoVE General

Direct Tracheal Instillation of Solutes into Mouse Lung


JoVE 1941 8/29/2010

1Department of Physiology, Emory University, 2Center for Respiratory Health, Emory University, 3Department of Medicine, Emory University

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

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 JoVE Neuroscience

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster


JoVE 50150 3/04/2013

1Institute of Genetics, University of Mainz, 2Department of Anatomy and Neuroscience, University of Melbourne

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

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 JoVE General

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury


JoVE 2079 8/04/2010

1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Department of Medicine and Genetics, Harvard Medical School

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

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 JoVE Neuroscience

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach


JoVE 3598 6/06/2012

School of Psychological Science, La Trobe University

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

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 JoVE Clinical and Translational Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract


JoVE 4016 11/28/2012

1St. Erik's Eye Hospital, Karolinska Institutet, 2Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

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 JoVE Neuroscience

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes


JoVE 1790 2/15/2010

STED Microscopy of Synaptic Function, European Neuroscience Institute Göttingen

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

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 JoVE General

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats


JoVE 2260 10/16/2010

1Department of Oncology, Georgetown University, 2Institute of Biomedicine, University of Turku Medical Faculty

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

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 JoVE General

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death


JoVE 4442 10/21/2012

Department of Molecular Microbiology, Faculty of Medicine, Technion - Israel Institute of Technology

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

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 JoVE Immunology and Infection

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells


JoVE 2195 12/18/2010

Department of Pathology and Immunology, Washington University School of Medicine

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

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 JoVE Applied Physics

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material


JoVE 50254 5/20/2013

1Department of Biomedical Science, Cornell University, 2Department of Ecology and Evolutionary Biology, Cornell University, 3Cornell University Museum of Vertebrates, 4Department of Computer Science, Cornell University

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

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 JoVE Clinical and Translational Medicine

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index


JoVE 3417 1/02/2012

1Department of Psychiatry, University of Geneva School of Medicine, 2Signal Processing Laboratory, École Polytechnique Fédérale de Lausanne, 3Department of Radiology, University Hospital Center and University of Lausanne, 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

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 JoVE General

Whole-Body Nanoparticle Aerosol Inhalation Exposures


JoVE 50263 5/07/2013

1Department of Physiology and Pharmacology, School of Medicine, West Virginia University, 2Center for Cardiovascular and Respiratory Sciences, West Virginia University, 3National Institute for Occupational Safety and Health

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

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 JoVE General

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development


JoVE 4035 6/07/2012

Department of Microbiology and Immunology, Loyola University Medical Center

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

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 JoVE General

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity


JoVE 1173 5/05/2009

Bioscience Division, High Content Analysis Research and Development, Millipore Inc

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

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 JoVE Clinical and Translational Medicine

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease


JoVE 4228 10/03/2012

1German Center for Neurodegenerative Diseases, DZNE, 2Laboratory of Functional Neurogenomics, Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

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 JoVE General

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells


JoVE 3854 6/21/2012

Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgfβ1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

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 JoVE Neuroscience

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System


JoVE 2123 10/26/2010

Institute of Anatomy, Technische Universität Dresden

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

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 JoVE General

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development


JoVE 2466 1/07/2011

1Department of Veterinary Science, University of Wisconsin, Madison, 2Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 3Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

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 JoVE Clinical and Translational Medicine

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity


JoVE 50077 1/10/2013

1Department of Physical Medicine and Rehabilitation, University of Texas Health Science Center at Houston, 2UTHealth Motor Recovery Laboratory, TIRR Memorial Hermann Hospital, 3The Institute of Rehabilitation and Research (TIRR), TIRR Memorial Hermann Hospital

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

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 JoVE General

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain


JoVE 1262 5/14/2009

1Department of Physiology, University of Montreal, 2Ecole d’optometrie, University of Montreal, 3Stereology Resource Center

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

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 JoVE Clinical and Translational Medicine

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen


JoVE 3586 5/30/2012

1Department of Chemistry and Biochemistry, University of Windsor, 2Chemistry Department and Centre for Biotechnology, Brock University

We have synthesized a novel analogue of pancratistatin with comparable anti-cancer activity as native pancratistatin; interestingly, combinatory treatment with tamoxifen yielded a drastic enhancement in apoptotic and autophagic induction by mitochondrial targeting with minimal effect on noncancerous fibroblasts. Thus, JCTH-4 in combination with tamoxifen could provide a safe anti-cancer therapy.

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