JoVE: Journal of Visualized Experiments

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• Transcranial Direct Current Stimulation (tDCS) in Mice

  Posted:Sun, 23 Sep 2018 15:00 EST

  Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

  

  

• Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding

  Posted:Sun, 23 Sep 2018 14:00 EST

  Here, we present a protocol for fabrication of 3D graphene-based polyhedrons via origami-like self-folding.

  

  

• The Combined Use of Transcranial Direct Current Stimulation and Robotic Therapy for the Upper Limb

  Posted:Sun, 23 Sep 2018 13:00 EST

  The combined use of transcranial direct current stimulation and robotic therapy as an add-on for conventional rehabilitation therapy may result in improved therapeutic outcomes due to modulation of brain plasticity. In this article, we describe the combined methods used in our institute for improving motor performance after stroke.

  

  

• Expression of Cementitious Pore Solution and the Analysis of Its Chemical Composition and Resistivity Using X-ray Fluorescence

  Posted:Sun, 23 Sep 2018 13:00 EST

  This protocol describes the procedure to express fresh pore solution from cementitious systems and the measurement of its ionic composition using X-ray fluorescence. The ionic composition can be used to calculate pore solution electrical resistivity, which can be used, together with concrete electrical resistivity, to determine the formation factor.

  

  

• An Available Technique for Preparation of New Cast MnCuNiFeZnAl Alloy with Superior Damping Capacity and High Service Temperature

  Posted:Sun, 23 Sep 2018 12:00 EST

  Here we present a protocol to obtain a novel Mn-Cu-based alloy with excellent comprehensive performances by a high-quality smelting technology and reasonable heat treatment methods.

  

  

• Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology

  Posted:Fri, 21 Sep 2018 14:00 EST

  We demonstrate a 24 h heart rate recording methodology to evaluate the influence of concussion across the recovery trajectory in youth athletes, within an ecologically valid context.

  

  

• Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

  Posted:Fri, 21 Sep 2018 13:00 EST

  We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

  

  

• A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts

  Posted:Thu, 20 Sep 2018 15:00 EST

  We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

  

  

• Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

  Posted:Thu, 20 Sep 2018 14:00 EST

  Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

  

  

• Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

  Posted:Thu, 20 Sep 2018 13:00 EST

  Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

  

  

• Anogenital Distance and Perineal Measurements of the Pelvic Organ Prolapse (POP) Quantification System

  Posted:Thu, 20 Sep 2018 12:00 EST

  This manuscript describes the procedures to perform anogenital distance (AGD) and perineal measurements standardized by the Pelvic Organ Prolapse Quantification System (POP-Q): perineal body (PB) and genital hiatus (GH). These measurements are compared in women with and without pelvic organ prolapse.

  

  

• An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

  Posted:Wed, 19 Sep 2018 15:00 EST

  Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

  

  

• Application of Design Aspects in Uniaxial Loading Machine Development

  Posted:Wed, 19 Sep 2018 14:00 EST

  Here we present a protocol to develop a pure uniaxial loading machine. Critical design aspects are employed to ensure accurate and reproducible testing results.

  

  

• Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

  Posted:Wed, 19 Sep 2018 13:00 EST

  Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

  

  

• Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

  Posted:Wed, 19 Sep 2018 13:00 EST

  Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

  

  

• Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

  Posted:Wed, 19 Sep 2018 12:00 EST

  Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

  

  

• Simultaneous Cryosectioning of Multiple Rodent Brains

  Posted:Tue, 18 Sep 2018 15:00 EST

  Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

  

  

• Negative Additive Manufacturing of Complex Shaped Boron Carbides

  Posted:Tue, 18 Sep 2018 14:00 EST

  A method called negative additive manufacturing is used to produce near fully dense complex shaped boron carbide parts of various length scales. This technique is possible via the formulation of a novel suspension involving resorcinol-formaldehyde as a unique gelling agent that leaves behind a homogenous carbon sintering aid after pyrolysis.

  

  

• Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

  Posted:Tue, 18 Sep 2018 14:00 EST

  Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

  

  

• Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

  Posted:Tue, 18 Sep 2018 14:00 EST

  Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

  

  

• Olfactory Context Dependent Memory: Direct Presentation of Odorants

  Posted:Tue, 18 Sep 2018 13:00 EST

  The use of an olfactometer for directly presenting odorants opens exciting opportunities for researchers of olfactory memory. The current paper discusses issues related to this methodology as related to a previously published experiment on olfactory context dependent memory.

  

  

• 'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake

  Posted:Tue, 18 Sep 2018 13:00 EST

  The 'Boden Food Plate' is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method.

  

  

• CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

  Posted:Tue, 18 Sep 2018 12:00 EST

  We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

  

  

• Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

  Posted:Sun, 16 Sep 2018 14:00 EST

  Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

  

  

• Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

  Posted:Sun, 16 Sep 2018 13:00 EST

  The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

  

  

• Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

  Posted:Sat, 15 Sep 2018 15:00 EST

  micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

  

  

• Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

  Posted:Sat, 15 Sep 2018 14:00 EST

  This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

  

  

• A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

  Posted:Sat, 15 Sep 2018 13:00 EST

  Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

  

  

• Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

  Posted:Fri, 14 Sep 2018 15:00 EST

  This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

  

  

• Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

  Posted:Fri, 14 Sep 2018 14:00 EST

  Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

  

  

• Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

  Posted:Fri, 14 Sep 2018 14:00 EST

  We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

  

  

• Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

  Posted:Fri, 14 Sep 2018 14:00 EST

  Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

  

  

• Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

  Posted:Fri, 14 Sep 2018 13:00 EST

  To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

  

  

• Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

  Posted:Fri, 14 Sep 2018 13:00 EST

  Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

  

  

• CRISPR-Mediated Reorganization of Chromatin Loop Structure

  Posted:Fri, 14 Sep 2018 12:00 EST

  Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

  

  

• Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

  Posted:Thu, 13 Sep 2018 15:00 EST

  Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

  

  

• 3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

  Posted:Thu, 13 Sep 2018 14:00 EST

  The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

  

  

• Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

  Posted:Thu, 13 Sep 2018 14:00 EST

  Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

  

  

• One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes

  Posted:Thu, 13 Sep 2018 14:00 EST

  Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching.

  

  

• Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

  Posted:Thu, 13 Sep 2018 13:00 EST

  This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

  

  

• A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

  Posted:Thu, 13 Sep 2018 13:00 EST

  Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

  

  

• Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

  Posted:Thu, 13 Sep 2018 12:00 EST

  Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

  

  

• Stereolithographic 3D Printing with Renewable Acrylates

  Posted:Wed, 12 Sep 2018 15:00 EST

  A protocol for additive manufacturing with renewable photopolymer resins on a stereolithography apparatus is presented.

  

  

• Chemoselective Preparation of 1-Iodoalkynes, 1,2-Diiodoalkenes, and 1,1,2-Triiodoalkenes Based on the Oxidative Iodination of Terminal Alkynes

  Posted:Wed, 12 Sep 2018 14:00 EST

  Herein, detailed protocols for the oxidative iodination of terminal alkynes using hypervalent-iodine reagents are presented, which chemoselectively afford 1-iodoalkynes, 1,2-diiodoalkenes, and 1,1,2-triiodoalkenes.

  

  

• Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

  Posted:Wed, 12 Sep 2018 14:00 EST

  Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

  

  

• In Situ Lithiated Reference Electrode: Four Electrode Design for In-operando Impedance Spectroscopy

  Posted:Wed, 12 Sep 2018 14:00 EST

  The incorporation of reference electrodes in a Li-ion battery provides valuable information to elucidate degradation mechanisms at high voltages. In this article, we present a cell design that accommodates multiple reference electrodes, along with the assembly steps to assure maximum accuracy of the data obtained in electrochemical measurements.

  

  

• Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

  Posted:Wed, 12 Sep 2018 13:00 EST

  Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

  

  

• Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

  Posted:Wed, 12 Sep 2018 13:00 EST

  Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

  

  

• An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

  Posted:Wed, 12 Sep 2018 12:00 EST

  In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

  

  

• Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

  Posted:Tue, 11 Sep 2018 15:00 EST

  Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

  

  

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