JoVE General
Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry
1Department of Neuroscience, Karolinska Institutet, 2National Institute on Drug Abuse (NIDA)
The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.
JoVE Clinical and Translational Medicine
Bronchial Thermoplasty: A Novel Therapeutic Approach to Severe Asthma
1Director of Pulmonary Special Procedures and the Lung Cancer Center, Virginia Hospital Center, 2Chief, Division of Pulmonary Critical Care and Sleep Medicine, Virginia Hospital Center
Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.
JoVE General
Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators
1Department of Anesthesia, Stanford University School of Medicine, 2Department of Anaesthesiology, University of Mannheim, 3Department of Anaesthesiology, University of Heidelberg
A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.
JoVE Neuroscience
F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes
Department of Internal Medicine, Yale University
A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.
JoVE General
Upright Imaging of Drosophila Embryos
1Department of Biology, Case Western Reserve University, 2Department of Genetics, Case Western Reserve University
Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.
JoVE General
Ex vivo Mechanical Loading of Tendon
1Department of Bioengineering, University of California, Berkeley, 2Department of Bioengineering, University of California, Berkeley; Division of Occupational Medicine, University of California, San Francisco
A new in vitro system for simultaneously loading four tendons in culture is described.
JoVE General
Determining heat and mechanical pain threshold in inflamed skin of human subjects
Department of Anesthesia, Stanford University School of Medicine
Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.
JoVE Bioengineering
Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University
The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.
JoVE General
Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye
Life Sciences Group, Hitachi High Technologies America
Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.
JoVE General
Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI
We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
JoVE Immunology and Infection
Plaque Assay for Murine Norovirus
Department of Microbiology and Immunology, University of Michigan, Ann Arbor
Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV’s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.
JoVE General
Ole Isacson: Development of New Therapies for Parkinson's Disease
McLean Hospital, Harvard Stem Cell Institute, Harvard Medical School
Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.
JoVE Immunology and Infection
ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza
InDevR, Inc.
ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.
JoVE Neuroscience
Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS
1Department of Zoology, University of Oklahoma - Norman, 2Department of Biology, Brandeis University
We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.
JoVE Clinical and Translational Medicine
Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas
1Division of Pediatric and Fetal Surgery, Department of Surgery, University of California, San Francisco, 2Department of Pathology, University of Alberta, 3Department of Obstretics and Gynecology, University of California, San Francisco, 4Department of Radiology, University of California, San Francisco
Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins’ circulations. Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.
JoVE Clinical and Translational Medicine
Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test
Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic
Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.
JoVE General
Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
1Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, 2Department of Internal Medicine, Washington University School of Medicine, 3Department of Pediatrics, Washington University School of Medicine
Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.
JoVE Clinical and Translational Medicine
Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)
1Dept. of Neurology, University Medicine Göttingen, 2DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany
A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.
JoVE Neuroscience
GABA-activated Single-channel and Tonic Currents in Rat Brain Slices
Department of Neuroscience, Uppsala University, Sweden
We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.
JoVE General
Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes
1Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biomedical Engineering and Cardiac Bioelectricity and Arrhythmia Center, Washington University in St. Louis
Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.
JoVE General
Gastrointestinal Motility Monitor (GIMM)
Department of Anatomy and Neurobiology, The University of Vermont
Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.
JoVE Neuroscience
Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging
1Center for the Neural Basis of Cognition, 2Department of Psychology, University of Pittsburgh, 3Department of Psychology, Carnegie Mellon University, 4Department of Bioengineering, University of Pittsburgh
We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.
JoVE General
Measurement of Lifespan in Drosophila melanogaster
1Department of Molecular and Integrative Physiology, University of Michigan, 2Cellular and Molecular Biology Program, University of Michigan
Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.
JoVE Clinical and Translational Medicine
Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye
1Division of Neonatology, Department of Pediatrics, Leiden University Medical Center, 2Division of Fetal Therapy, Department of Obstetrics, Leiden University Medical Center, 3Department of Obstetrics, Leiden University Medical Center
Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.
JoVE Neuroscience
Laser Capture Microdissection of Drosophila Peripheral Neurons
1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.
JoVE Neuroscience
Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI
Department of Psychology, University of Alabama at Birmingham
A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.
JoVE General
Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy
The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.
JoVE Clinical and Translational Medicine
Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)
Division of Gastroenterology, Washington University School of Medicine
We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.
JoVE General
Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting
1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.
JoVE General
In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion
Department of Biochemistry, University of Nebraska, Lincoln
The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.
JoVE General
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.
JoVE Bioengineering
Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, 2Department of Pharmacology, University of Pennsylvania
This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).
JoVE Neuroscience
Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation
1Division of Molecular Neurobiology, MRC National Institute for Medical Research, 2Confocal and Image Analysis Laboratory, National Institute for Medical Research, 3Physiopathologie de la plasticité neuronale, Neurocentre Magendie, Université de Bordeaux
This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.
JoVE Neuroscience
Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats
Department of Neuroscience, University of Lethbridge
Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.
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