Hebrew University View Institution's Website 18 articles published in JoVE Neuroscience Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors Rita Gutorov1, Ben Katz1, Baruch Minke1 1Department of Medical Neurobiology, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Hebrew University We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems. Developmental Biology Fluorimetric Techniques for the Assessment of Sperm Membranes Alisa Komsky-Elbaz1,2, Zvi Roth1,2 1Animal Sperm Research Center, Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, 2Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented. Medicine Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD) George M. Solomon1, Inez Bronsveld2, Kathryn Hayes3, Michael Wilschanski4, Paola Melotti5, Steven M. Rowe1, Isabelle Sermet-Gaudelus6,7 1Department of Medicine and the Gregory Fleming James Cystic Fibrosis Center, University of Alabama at Birmingham, 2Department of Pulmonology and Tuberculosis, University Medical Center Utrecht, 3Center for Experimental Medicine, Queens University, Northern Ireland, 4Hadassah Hebrew University Medical Center, Jerusalem, 5Centro Fibrosi Cistica, Azienda Ospedaliera Universitaria Integrata, 6Service de Pneumologie et Allergologie Pédiatriques and Center de Ressources et de Compétence de la Mucoviscidose, Hôpital Necker Enfants Malades, 7INSERM U 1151, Institut Necker Enfants Malades 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. Neuroscience Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors Ben Katz*1, Rita Gutorov*1, Elisheva Rhodes-Mordov1, Roger C. Hardie2, Baruch Minke1 1Department of Medical Neurobiology, Faculty of Medicine and the Edmond and Lily Safra Center for Brain Sciences (ELSC), Hebrew University, 2Department of Physiology, Development and Neuroscience, University of Cambridge Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel. Genetics Controllable Ion Channel Expression through Inducible Transient Transfection Matan Geron*1, Adina Hazan*1, Avi Priel1 1The Institute for Drug Research (IDR), School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem (HUJI) Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter. Medicine Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model Assaf Dotan1, Israel Kremer1,2, Orly Gal-Or1, Tami Livnat3, Arie Zigler4, Dan Bourla1, Dov Weinberger1,2,3 1Department of Ophthalmology, Rabin Medical Center, Beilinson Campus, 2Sackler Faculty of Medicine, Tel Aviv University, 3Laboratory of Eye Research, Felsenstein Medical Research Center, 4Racah Institute of Physics, The Hebrew University We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy). Immunology and Infection Isolation and Characterization of Neutrophils with Anti-Tumor Properties Ronit Vogt Sionov1, Simaan Assi1, Maya Gershkovitz1, Jitka Y. Sagiv1, Lola Polyansky1, Inbal Mishalian2, Zvi G. Fridlender2, Zvi Granot1 1Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University Medical School, 2Institute of Pulmonary Medicine, Hadassah-Hebrew University Medical Center Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo. Engineering Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds Ori Hazut1,2, Arunava Agarwala1,2, Thangavel Subramani1,2, Sharon Waichman1,2, Roie Yerushalmi1,2 1Institute of Chemistry, The Hebrew University of Jerusalem, 2Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem A detailed procedure for surface doping of Silicon interfaces is provided. The ultra-shallow surface doping is demonstrated by using phosphorus containing monolayers and rapid annealing process. The method can be used for doping of macroscopic area surfaces as well as nanostructures. Chemistry Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay Nitzan Ganot*1, Sigalit Meker*1, Lilia Reytman*1, Avia Tzubery*1, Edit Y. Tshuva1 1Institute of Chemistry, The Hebrew University of Jerusalem A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay. Behavior Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome Shahar Barbash1, Geula Hanin1, Hermona Soreq1 1Department of Biological Chemistry and The Edmond & Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem 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. Neuroscience Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo Ayelet Kohl1, Yoav Hadas2, Avihu Klar2, Dalit Sela-Donenfeld1 1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, 2Department of Medical Neurobiology, The Hebrew University of Jerusalem 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. Biology 4D Imaging of Protein Aggregation in Live Cells Rachel Spokoini*1, Maya Shamir*1, Alma Keness*1, Daniel Kaganovich1 1Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy. Biology Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter Oswald J. Schmitz1, Mark A. Bradford1, Michael S. Strickland1,2, Dror Hawlena3 1School of Forestry and Environmental Studies, Yale University, 2Department of Biological Sciences, Virginia Tech, 3Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes. Chemistry LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations Ido Braslavsky1,2, Ran Drori1 1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner. Bioengineering High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs Felix Moltzahn1,2,3, Nathan Hunkapiller1,2,4, Alain A. Mir5, Tal Imbar1,2,6, Robert Blelloch1,2,3,7 1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Center for Reproductive Sciences, University of California San Francisco, 3Department of Urology, University of California San Francisco, 4Department of Cell and Tissue Biology, University of California San Francisco, 5Fluidigm Corporation, Fluidigm Corporation, 6Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, 7UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample. Biology Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells Malka Nissim-Rafinia1, Eran Meshorer1 1Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells. Biology Single Cell Transfection in Chick Embryos Raz Ben-Yair1, Chaya Kalcheim1 1Department of Medical Neurobiology, Hadassah Medical School - Hebrew University Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations. Biology Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation Oshri Avraham*1, Sophie Zisman*1, Yoav Hadas1, Lilach Vald1, Avihu Klar1 1Department of Medical Neurobiology, Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.