JoVE Editorial
June 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Back in 1905, in what is now the Czech Republic, Eduard Zirm performed the first corneal transplantation surgery (keratoplasty), which restored vision to a patient blinded by corneal injury. Today, eye banks all over the world prepare, store, and distribute donated corneas to hospitals so that thousands of sight-saving keratoplasties can be performed every year. In June 2012, JoVE has its eye on two research groups, one from Italy and the other from Michigan, who demonstrate two distinct methods for corneal graft preparation prior to transplantation.
JoVE Clinical and Translational Medicine
A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe
1Fondazione Banca Degli Occhi del Veneto O.N.L.U.S., 2Telethon Institute for Genetics & Medicine (T.I.G.E.M.)
The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.
JoVE General
Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles
Plant Mycotoxin Research, U.S. Department of Agriculture, Agricultural Research Service
A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.
JoVE Clinical and Translational Medicine
Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET
1Department of Internal Medicine D, Experimental Nephrology, University of Münster, 2Department of Nuclear Medicine, University of Münster, 3European Institute for Molecular Imaging, University of Münster
We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.
JoVE Immunology and Infection
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
1Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, 2Department of Immunoregulation, Helmholtz Center for Infection Research
We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).
JoVE Clinical and Translational Medicine
Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction
1Institute for Laboratory Animal Science and Experimental Surgery, RWTH-Aachen University, 2Department of Hepato-Biliary-Pancreatic Surgery and Transplantation, Graduate School of Medicine, Kyoto University
Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.
JoVE Immunology and Infection
Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Department of Microbiology and Molecular Genetics, University of California, Irvine
Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.
JoVE General
Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector
1Center for Regenerative Medicine (CReM), Boston University School of Medicine, 2Department of Hematology, Children's Hospital of Philadelphia, 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia
Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.
JoVE General
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.
JoVE Immunology and Infection
TransFLP — A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination
Global Health Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL)
A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.
JoVE Clinical and Translational Medicine
Murine Bioluminescent Hepatic Tumour Model
1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, 2Department of Computer Science, University College Cork, 3South Infirmary Victoria University Hospital
This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.
JoVE Clinical and Translational Medicine
Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology
1Department of Surgery, Division of Surgical Oncology, University Medical Center Groningen, 2Helmholtz Zentrum, Technical University Munich, 3Department of Obstetrics and Gynaecology, University Medical Center Groningen
Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.
JoVE General
A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
1Department of Microbiology, Medical School, University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University of Minnesota
A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.
JoVE General
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)
Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.
JoVE General
Molecular Evolution of the Tre Recombinase
Max Plank Institute for Molecular Cell Biology and Genetics, Dresden
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.
JoVE Neuroscience
Double Fluorescence in situ Hybridization in Fresh Brain Sections
1Department of Brain and Cognitive Sciences, University of Rochester, 2Center for Visual Science, University of Rochester
This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.
JoVE General
Assay for Neural Induction in the Chick Embryo
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.
JoVE Clinical and Translational Medicine
The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage
Department of Surgery, Division of Abdominal Surgery, University Medical Center Groningen
The C-seal is a biofragmentable drain protecting the stapled colorectal anastomosis. In this video, details of the manufacturing, deployment and use are shown.
JoVE General
Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos
Department of Genetics, University of Georgia
This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.
JoVE Editorial
October 2011: This Month in JoVE
Here are some highlights from the October 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE Applied Physics
Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy
Materials Sciences Division, Lawrence Berkeley National Laboratory
We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.
JoVE General
Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis
Department of Biology, Portland State University
We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.
JoVE General
Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development
Department of Orthopaedic Surgery, University of California San Francisco
This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.
JoVE Applied Physics
Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells
1Center for Innovative Fuel Cells and Battery Technologies, School of Materials Science and Engineering, Georgia Institute of Technology, 2School of Chemistry and Biochemistry, Georgia Institute of Technology
We present a unique platform for characterizing electrode surfaces in solid oxide fuel cells (SOFCs) that allows simultaneous performance of multiple characterization techniques (e.g. in situ Raman spectroscopy and scanning probe microscopy alongside electrochemical measurements). Complementary information from these analyses may help to advance toward a more profound understanding of electrode reaction and degradation mechanisms, providing insights into rational design of better materials for SOFCs.
JoVE Clinical and Translational Medicine
Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure
1Program in Developmental Biology, Children's Memorial Research Center, 2Department of Pediatrics, Northwestern University
In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.
JoVE General
Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos
1Institut de Recherches Cliniques de Montréal (IRCM), 2Department of Biochemistry, Université de Montréal
Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.
JoVE Clinical and Translational Medicine
Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
JoVE Applied Physics
A Method to Fabricate Disconnected Silver Nanostructures in 3D
1School of Engineering and Applied Sciences, Harvard University, 2Department of Physics, Harvard University
Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.
JoVE General
In Situ Hybridization for the Precise Localization of Transcripts in Plants
The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.
JoVE General
Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development
1Department of Biological Sciences, The George Washington University, 2Department of Anatomy and Regenerative Biology, The George Washington University
The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.
JoVE Immunology and Infection
Estimating Virus Production Rates in Aquatic Systems
Department of Microbiology, University of Tennessee
The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.
JoVE Immunology and Infection
Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory
A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.
JoVE General
Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton
1Biological Sciences, Kent State University, 2Marine Sciences, University of Georgia (UGA)
Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.
JoVE General
Dissection of the Adult Zebrafish Kidney
Department of Biological Sciences, University of Notre Dame
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
JoVE Neuroscience
Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles
Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania
This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.
JoVE General
Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts
Department of Molecular and Cellular Biology, University of Guelph
In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.
JoVE General
In situ Protocol for Butterfly Pupal Wings Using Riboprobes
1Department of Biological Sciences, SUNY-University at Buffalo, 2Dept. Ecology and Evolutionary Biology, Yale University
In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.
JoVE Immunology and Infection
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University
This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).
JoVE Clinical and Translational Medicine
An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery
1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine
An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.
JoVE General
Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model
Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.
JoVE Neuroscience
Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology
1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc.
A combination of different techniques to maximize data collection from mouse tissue is presented.
JoVE General
Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay
Medical Research Council, Clinical Sciences Centre, Imperial College, Hammersmith Hospital
Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.
JoVE General
Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Department of Molecular, Cellular and Developmental Biology, Yale University
Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.
JoVE General
Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer
1Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Tucson, 2Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, 3Department of Surgery, College of Medicine, University of Arizona, Tucson, 4Biomedical Diagnostics and Research, Tucson, AZ, 5Department of Medicine, College of Medicine, University of Arizona, Tucson
Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.
JoVE Clinical and Translational Medicine
Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer
1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine
We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.
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 Editorial
December 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the December 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs
Department of Biological Sciences, Union College
By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.
JoVE General
OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides
1Energy Biosciences Institute, University of California, Berkeley, 2Department of Plant and Microbial Biology, University of California, Berkeley
A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.
JoVE Bioengineering
Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)
1Applied Ocean Physics and Engineering, Woods Hole Oceanographic Institution, 2Environmental Science and Marine Biology, Roger Williams University, 3Marine Biology Laboratory, Whitman Center, 4Department of Biology, Providence College, 5Departments of Aeronautics and Bioengineering, California Institute of Technology
This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.
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