Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates
We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics.
Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model
1Department of Obstetrics, Perinatal Pharmacology, University Hospital Zurich, 2Laboratory for Materials - Biology Interactions, EMPA Swiss Federal Laboratories for Materials Testing and Research, 3Graduate School for Cellular and Biomedical Sciences, University of Bern
The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.
1Department of Biomedical Engineering, University of Southern California, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Geffen School of Medicine, University of California, Los Angeles
This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.
1Programa de Pós-Graduação em Ciências Médica, Universidade Federal do Rio Grande do Sul, 2Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), 3Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 4De Montfort University
Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has shown initial therapeutic effects in several neurological conditions. The main mechanism underlying these therapeutic effects is the modulation of cortical excitability. Therefore, online monitoring of cortical excitability would help guide stimulation parameters and optimize its therapeutic effects. In the present article we review the use of a novel device that combines simultaneous tDCS and EEG monitoring in real time.
1Department of Psychology, University of Cincinnati, 2Department of Neurology, University of Cincinnati
In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.
The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.
A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics
1Brain Tumor Research Centre, Hospital for Sick Children, Toronto Medical Discovery Tower, 2Ontario Cancer Institute, Princess Margaret Hospital, 3Neurosurgery, Toronto Western Hospital
We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.
Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells
1Pediatric Diabetes Research Center, University of California, San Diego, 2Janssen Research & Development, 3Department of Medicine, University of California, San Diego
Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method—adapted from a coculture model of synaptogenesis—for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.
Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Graduate Program in Quantitative and Computational Biology, Princeton University, 3Department of Molecular Biology, Princeton University
This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.
The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.
Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.
An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy
1Discipline of Biomedical Science, School of Medical Sciences, Sydney Medical School, University of Sydney, 2School of Biomedical Sciences and Pharmacy, University of Newcastle
Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.
Cell Signaling Technology, Inc.
Here we provide a comprehensive western blot protocol developed and used at Cell Signaling Technology that includes optimal reagents and support information to ensure optimal results.
Cell Signaling Technology, Inc.
Here we provide an extensive western blot troubleshooting guide based on our many years of experience, offering solutions to save you valuable time and reagents.
Department of Cardiology, University of Heidelberg
Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.
1Dynamique des Interactions Hôte Pathogène, Institut Pasteur, Paris, France, 2Imagopole, Institut Pasteur, Paris, France, 3Pathogenomique Mycobacterienne Integrée, Institut Pasteur, Paris, France
We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.
High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots
1Application Development, Akonni Biosystems, Inc., 2Manufacturing, Akonni Biosystems, Inc., 3Engineering, Akonni Biosystems, Inc., 4Research & Development, Akonni Biosystems, Inc.
TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.
1Department of Biomedical Engineering, University at Buffalo, The State University of New York, 2Department of Creative IT Engineering, Pohang University of Science and Technology (POSTECH), 3School of Electrical Engineering and Computer Science, Kyungpook National University
Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.
Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior
1Department of Oral and Maxillofacial Surgery, University of Florida College of Dentistry, 2Department of Neuroscience, McKnight Brain Institute, University of Florida College of Medicine, 3Stoelting Co., 4Department of Orthodontics, University of Florida
We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.
1Biomedical Sciences, Tufts University Cummings School of Veterinary Medicine, 2School of Healthcare Sciences, Manchester Metropolitan University
This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.
Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome
MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.
Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies
1Department of Neuroscience, University of Turin, 2Neuroscience Institute of Turin (NIT), University of Turin, 3Neuroscience Institute Cavalieri-Ottolenghi (NICO), University of Turin, 4Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin
Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.
In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury
1Department of Surgery, Royal Infirmary of Edinburgh, 2Department of Nephrology, Royal Infirmary of Edinburgh
Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.
We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.
In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGCα1-/- mice using pressure myography.
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the June 2013 Issue of Journal of Visualized Experiments (JoVE).
1Department of Psychology, University of Wisconsin-Milwaukee, 2McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, 3Department of Neurology, Medical College of Wisconsin
This article describes how to record amygdala activity with magnetoencephalography (MEG). In addition this article will describe how to conduct trace fear conditioning without awareness, a task that activates the amygdala. It will cover 3 topics: 1) Designing a trace conditioning paradigm using backward masking to manipulate awareness. 2) Recording brain activity during the task using magnetoencephalography. 3) Using source imaging to recover signal from subcortical structures.
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Investigation of the Uncanny Valley Hypothesis and affective experience requires an understanding of the hypothesis' dimension of human likeness (DHL). This protocol allows representation of the DHL and examination of categorical perception. Use of the same stimuli and fMRI to distinguish brain regions responsive to physical and category change is illustrated.
Simultaneous electroencephalography (EEG) and functional Magnetic Resonance imaging (fMRI) is a powerful neuroimaging tool. However, the inside of an MRI scanner forms a difficult environment for EEG data recording and safety must be considered whenever operating EEG equipment inside a scanner. Here, we present an optimised EEG-fMRI data acquisition protocol.
This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.
Mice can swim, but many strains appear to find this activity stressful. To overcome this problem mazes have been devised where escape from shallow water is used to motivate behaviour. These have been demonstrated to support learning at least as good as the traditional and widely used Morris water maze.
Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.
Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation
We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.
Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein
The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.
High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.
Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages
1Department of Biological Sciences, Kingsborough Community College, 2Institute for Cellular and Molecular Biology, University of Texas at Austin, 3New Jersey Center for Science, Technology and Mathematics, Kean University
We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.
CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.
Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo
How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox–plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.
Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.
A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.
1Cancer Prevention and Control Program, Fox Chase Cancer Center, 2Department of Psychiatry, University of Pennsylvania, 3Department of Psychology, Drexel University, 4Department of Medicine, Fox Chase Cancer Center, 5Cancer Prevention and Control Program, The Cancer Institute of New Jersey
This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.
We describe a method for in-situ tapering of As2S3 fibers to achieve efficient mid-infrared supercontinuum generation. By tapering while monitoring the supercontinuum’s spectrum, the spectral width can be maximized for a fiber taper. In-situ fiber tapering can be applied to optimize the performance of other fiber-based devices.
Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution
1Institute of Biophysics, National Research Council of Italy, 2Dipartimento di Sistemi e Informatica, Università di Firenze, 3Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia
We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.
Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.
An approach to neural network modeling on the LEGO Mindstorms robotics platform is presented. The method provides a simulation tool for invertebrate neuroscience research in both the research lab and the classroom. This technique enables the investigation of biomimetic robot control principles.
Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.
Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.
Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides
In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.
A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided
Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.