JoVE Neuroscience
Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones
Department of Neuroscience, University of Minnesota
A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.
JoVE Neuroscience
Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation
1Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center
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.
JoVE General
Single-cell Suction Recordings from Mouse Cone Photoreceptors
We will show how to record flash responses from single mouse cones using a suction electrode.
JoVE Neuroscience
Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine
We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.
JoVE Neuroscience
Mechanical Manipulation of Neurons to Control Axonal Development
Department of Zoology, Michigan State University, East Lansing
Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.
JoVE Neuroscience
Neural Explant Cultures from Xenopus laevis
Department of Cell Biology, Harvard Medical School
Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.
JoVE General
Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones
Department of Biological Sciences, Purdue University
Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.
JoVE Neuroscience
Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging
Storm Eye Institute, Medical University of South Carolina
A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.
JoVE Clinical and Translational Medicine
Multifocal Electroretinograms
John A. Moran Eye Center, University of Utah
The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.
JoVE Clinical and Translational Medicine
Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography
1Carestream Molecular Imaging, 2Department of Chemistry and Biochemistry, University of Notre Dame, 3Freimann Life Science Center, University of Notre Dame, 4Research and Development, Oncovision, GEM-Imaging S.A.
Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.
JoVE Bioengineering
Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip
1Department of Plant Biology, Carnegie Institution for Science, 2Howard Hughes Medical Institute, 3Departments of Applied Physics and Bioengineering, Stanford University, 4Department of Microsystems Engineering (IMTEK) and Center for Biological Signaling Studies (BIOSS), University of Freiburg
This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.
JoVE Clinical and Translational Medicine
Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer
1Watson School of Biological Sciences, 2Cold Spring Harbor Laboratory, 3Departments of Medical Genetics, University of Oslo and Oslo University Hospital
We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.
JoVE Neuroscience
In vivo Laser Axotomy in C. elegans
A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.
JoVE General
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
1Department of Evolutionary Functional Genomics, Evolutionary Biology Center, Uppsala University, 2Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences
We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.
JoVE Neuroscience
DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord
Developmental Neurobiology, Max Delbrück Center for Molecular Medicine
The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.
JoVE Immunology and Infection
Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children
1Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland, 2University of Basel, Basel, Switzerland
Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.
JoVE Bioengineering
Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans
Department of Biology, University of Utah
Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.
JoVE General
An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species
Warnell School of Forestry and Natural Resources, University of Georgia (UGA)
Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.
JoVE General
Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos
1Genetics Training Program, University of Wisconsin-Madison, 2Department of Anatomy, University of Wisconsin-Madison, 3Department of Zoology, University of Wisconsin-Madison, 4Cell and Molecular Biology Training Program, University of Wisconsin-Madison
This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.
JoVE Clinical and Translational Medicine
Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme
Department of Radiation Oncology, University of Pennsylvania
We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.
JoVE Neuroscience
Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation
Department of Pathology and Immunology, Washington University School of Medicine
This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.
JoVE Neuroscience
Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
Department of Anatomy, University of Wisconsin-Madison
This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.
JoVE General
Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions
Department of Biology, Brandeis
This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.
JoVE General
In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina
1Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine, 2Department of Biological Sciences, University of Notre Dame, 3Center for Zebrafish Research, University of Notre Dame
A method to conditionally knockdown a target protein’s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein’s role in the regenerating or intact retina.
JoVE Neuroscience
Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons
1Department of Neuroscience, Ohio State University College of Medicine, 2Department of Ophthalmology and Visual Science, University of Texas Medical School
An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.
JoVE Immunology and Infection
In vivo Imaging of Transgenic Leishmania Parasites in a Live Host
1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa
An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.
JoVE Immunology and Infection
Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice
1Department of Microbiology and Immunology, University of Melbourne, 2Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen Medical Centre, 3The Centre for Dynamic Imaging, The Walter and Eliza Hall Institute for Medical Research
A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.
JoVE Immunology and Infection
Using Luciferase to Image Bacterial Infections in Mice
Microbial & Molecular Pathogenesis, Texas A&M Health Science Center
Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.
JoVE Neuroscience
Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.
JoVE General
Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons
Department of Biological Sciences, Simon Fraser University
Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.
JoVE Neuroscience
Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.
JoVE Clinical and Translational Medicine
X-ray Dose Reduction through Adaptive Exposure in Fluoroscopic Imaging
We are developing a dynamic adaptive exposure technique using our scanning beam digital X-ray system. Rather than exposing an object uniformly, the exposure is adapted depending on the opacity of the object. Here we show an experiment on an anthropomorphic phantom that resulted in a dose saving of 30%.
JoVE Neuroscience
Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures
1Department of Physiology, David Geffen School of Medicine at UCLA, 2Natural Science Division, Pepperdine University
This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.
JoVE Neuroscience
Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue
Department of Biology, College of William and Mary
Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.
JoVE General
Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly
1Department of Brain and Cognitive Science, MIT - Massachusetts Institute of Technology, 2Picower Institute for Learning and Memory, MIT - Massachusetts Institute of Technology
In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.
JoVE General
Micro-drive Array for Chronic in vivo Recording: Drive Fabrication
1Picower Institute for Learning and Memory, MIT - Massachusetts Institute of Technology, 2Department of Brain and Cognitive Science, MIT - Massachusetts Institute of Technology
In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.
JoVE Bioengineering
On-Chip Endothelial Inflammatory Phenotyping
Department of Biomedical Engineering, University of California, Davis
Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.
JoVE Bioengineering
High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT
1Department of Molecular Genetics, Weizmann Institute of Science, 2Department of Biological Regulation, Weizmann Institute of Science, 3Department of Chemical Infrastructure, Weizmann Institute of Science
Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).
JoVE General
Analyzing Large Protein Complexes by Structural Mass Spectrometry
Department of Biological Chemistry, Weizmann Institute of Science
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
JoVE Clinical and Translational Medicine
Pseudofracture: An Acute Peripheral Tissue Trauma Model
1Department of Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Aachen Medical Center
Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.
JoVE Immunology and Infection
Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse
Center for Molecular Imaging (CMI), University of Texas Health Science Center-Houston
Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.
JoVE General
Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry
Department of Chemistry, George Washington University
Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.
JoVE General
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Department of Biological Chemistry, Weizmann Institute of Science
Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.
JoVE General
Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method
Cardiovascular Research Center, University of Kentucky
The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.
JoVE Clinical and Translational Medicine
Angiogenesis in the Ischemic Rat Lung
The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.
JoVE Bioengineering
Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins
Biomaterials Research Group, University of Bayreuth
Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.
JoVE Neuroscience
Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI
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.
JoVE Bioengineering
Axon Stretch Growth: The Mechanotransduction of Neuronal Growth
1Departments of Biomedical Engineering, New Jersey Institute of Technology, 2Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey
A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.
JoVE Immunology and Infection
An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses
1Penn Ovarian Cancer Research Center, Center for Research on Reproduction and Womans Health, Department of Obstetrics and Gynecology, University of Pennsylvania-School of Medicine, 2Women's Cancer Program, Fox Chase Cancer Center
To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.
JoVE General
Dissection of Larval CNS in Drosophila Melanogaster
Department of Molecular Biology, Princeton University
In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.
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