Yale University View Institution's Website 61 articles published in JoVE Neuroscience Simultaneous Data Collection of fMRI and fNIRS Measurements Using a Whole-Head Optode Array and Short-Distance Channels Sara Sanchez-Alonso1, Rebecca R. Canale2, Isabel F. Nichoson1, Richard N. Aslin1,2,3 1Child Study Center, Yale University School of Medicine, 2Department of Psychology, University of Connecticut, 3Department of Psychology, Yale University We present a method for simultaneously collecting fMRI and fNIRS signals from the same subjects with whole-head fNIRS coverage. The protocol has been tested with three young adults and can be adapted for data collection for developmental studies and clinical populations. Bioengineering An Intra-Tissue Radiometry Microprobe for Measuring Radiance In Situ in Living Tissue Amanda L. Holt1,3, Yakir Luc Gagnon2,4, Alison M. Sweeney1,3 1Department of Physics, Yale University, 2Lund Vision Group, Department of Biology, Lund University, 3Department of Physics and Astronomy, University of Pennsylvania, 4Department of Biology, Duke University In this paper, a method for measuring radiance in situ in living tissue is described. This work includes details of the construction of micro-scale probes for different measurements of radiance and irradiance, provides guidance for mounting tissue for the characterization of radiance, and outlines computational methods for analyzing the resulting data. Neuroscience Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain Sofia Massaro Tieze1,2,3, Sreeganga S. Chandra1,2, D. J. Vidyadhara1,2 1Department of Neurology, Yale University, 2Department of Neuroscience, Yale University, 3Interdepartmental Neuroscience Program, Yale University This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution. Bioengineering In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles Sheng Chen1, Zachary Gao Sun2,3, Michael P. Murrell1,2,3 1Department of Biomedical Engineering, Yale University, 2Systems Biology Institute, Yale University, 3Department of Physics, Yale University In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane. Medicine Determining Binding Affinity (KD) of Radiolabeled Antibodies to Immobilized Antigens Erika Belitzky1, Alessandra Cavaliere1, Khashayar Rajabimoghadam1, Bernadette Marquez-Nostra1 1Department of Radiology and Biomedical Imaging, Yale University Here, a method is described to determine the binding affinity (KD) of radiolabeled antibodies to immobilized antigens. KD is the equilibrium dissociation constant that can be determined from a saturation binding experiment by measuring the total, specific, and nonspecific binding of a radiolabeled antibody at various concentrations to its antigen. Biochemistry Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins Yazgan Tuna1, Amer Al-Hiyasat1, Jonathon Howard1,2 1Department of Molecular Biophysics & Biochemistry, Yale University, 2Department of Physics, Yale University We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins. Bioengineering Engineered Lung Tissues Prepared from Decellularized Lung Slices Katherine L. Leiby1,2, Ronald Ng1, Stuart G. Campbell1,3, Laura E. Niklason1,4 1Department of Biomedical Engineering, Yale University, 2Yale School of Medicine, 3Department of Cellular and Molecular Physiology, Yale School of Medicine, 4Department of Anesthesiology, Yale School of Medicine This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells. Biochemistry In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays Ruchir C. Bobde*1,2, Ketul Saharan*1,2, Somanath Baral1,3, Surajit Gandhi1,2, Archana Samal1,2, Rajivgandhi Sundaram1, Ashish Kumar1,4, Ajit K. Singh1,5, Aritreyee Datta1, Dileep Vasudevan1 1Institute of Life Sciences, 2Regional Centre for Biotechnology, 3School of Biotechnology, KIIT University, 4Department of Molecular Biophysics and Biochemistry, Yale University, 5Department of Pharmacology, University of Vermont College of Medicine This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro. Biology Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy Nikola Lukic*1, Trishna Saha*1, Stefanie Lapetina1, Michal Gendler1, Gilad Lehmann1, Anthony J. Koleske2,3, Hava Gil-Henn1 1The Azrieli Faculty of Medicine, Bar-Ilan University, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Neuroscience, Yale University This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells. Neuroscience Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine Robert J. Wickham1, Makenzie Lehr*1, Lauryn Mitchell*1, Nii A. Addy2,3 1Department of Psychology, Elizabethtown College, 2Department of Psychiatry, Yale University, 3Department of Cellular and Molecular Physiology, Yale University The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release. Immunology and Infection Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System David Chetrit1, Donghyun Park1,2, Bo Hu3, Jun Liu1,2, Craig R. Roy1 1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions. Genetics Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting Lucas D. Sanor1, G. Parker Flowers1, Craig M. Crews1 1Department of Molecular, Cellular, and Developmental Biology, Yale University This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques. Biology Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture Laurel R. Yohe*1,2, Paolo Devanna*3, Kalina T.J. Davies4, Joshua H.T. Potter4, Stephen J. Rossiter4, Emma C. Teeling5, Sonja C. Vernes*3,6, Liliana M. Dávalos*2,7 1Department of Geology & Geophysics, Yale University, 2Department of Ecology & Evolution, Stony Brook University, 3Neurogenetics of Vocal Communication, Max Planck Institute for Psycholinguistics, 4School of Biological and Chemical Sciences, Queen Mary University of London, 5School of Biology & Environmental Science, University College Dublin, 6Donders Institute for Brain, Cognition and Behavior, 7Consortium for Inter-Disciplinary Environmental Research, Stony Brook University This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue. Bioengineering Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules Mohammed Mahamdeh1,2, Jonathon Howard1 1Department of Molecular Biophysics and Biochemistry, Yale University, 2Harvard Medical School, Harvard University This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays. Medicine Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis Toshihiko Isaji1,2,3, Shun Ono1,2,4,5, Takuya Hashimoto1,2,3, Kota Yamamoto1,2,3, Ryosuke Taniguchi1,2,3, Haidi Hu1,2, Tun Wang1,2, Jun Koizumi4, Toshiya Nishibe5, Katsuyuki Hoshina3, Alan Dardik1,2,6 1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, University of Tokyo, 4Department of Diagnostic Radiology, Tokai University School of Medicine, 5Department of Cardiovascular Surgery, Tokyo Medical University, 6Department of Vascular Surgery, VA Connecticut Healthcare Systems An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis. Bioengineering 3D Analysis of Multi-cellular Responses to Chemoattractant Gradients Tae-Yun Kang1, David Ellison2, Sung Hoon Lee1, Andrew J. Ewald2,3, Andre Levchenko1 1Department of Biomedical Engineering and Yale Systems Biology Institute, Yale University, 2Department of Biomedical Engineering, Johns Hopkins University, 3Center for Cell Dynamics and Department of Cell Biology, Johns Hopkins University We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs. Neuroscience A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum Regina T. Martuscello1, Elan D. Louis2,3,4, Phyllis L. Faust1 1Department of Pathology and Cell Biology, Columbia University, 2Division of Movement Disorders, Department of Neurology, Yale University, 3Department of Chronic Disease Epidemiology, Yale School of Public Health, Yale University, 4Center for Neuroepidemiology and Clinical Neurological Research, Yale School of Medicine, Yale University This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing. Bioengineering Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture Amanda S. Pellowe1, Holly M. Lauridsen1, Rita Matta1, Anjelica L. Gonzalez1 1Biomedical Engineering, Yale University Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices. Medicine Patch Angioplasty in the Rat Aorta or Inferior Vena Cava Hualong Bai1,2,3,4,5, Xin Li6, Takuya Hashimoto1,2,5, Haidi Hu1,2,5, Trenton R. Foster1,2,5, Jesse J. Hanisch1,2,5, Jeans M. Santana1,2,5, Alan Dardik1,2,5 1Department of Surgery, Yale University, 2Vascular Biology and Therapeutics Program, Yale University, 3Department of Vascular Surgery, First Affiliated Hospital of Zhengzhou University, 4Basic Medical College of Zhengzhou University, 5VA Connecticut Healthcare Systems, West Haven, CT, 6Department of Vascular Surgery, Xiangya Second Hospital of Central South University, Changsha, China We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation. Behavior Ultrasound Images of the Tongue: A Tutorial for Assessment and Remediation of Speech Sound Errors Jonathan L. Preston1,2, Tara McAllister Byun3, Suzanne E. Boyce2,4, Sarah Hamilton4, Mark Tiede2, Emily Phillips2, Ahmed Rivera-Campos4, Douglas H. Whalen2,5,6 1Department of Communication Sciences and Disorders, Syracuse University, 2Haskins Laboratories, 3Department of Communicative Sciences and Disorders, New York University, 4Department of Communication Sciences and Disorders, University of Cincinnati, 5Program in Speech-Language-Hearing Sciences, City University of New York Graduate Center, 6Department of Linguistics, Yale University Ultrasound imaging can be used to display the shape and movements of the tongue in real time during speech. The images can be used to determine the nature of speech sound errors. Visual feedback of the tongue can be used to facilitate improvements in speech sound production in clinical populations. Neuroscience SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy Joerg Nikolaus1,2, Erdem Karatekin1,2,3,4 1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS) Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured. Neuroscience A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice Melanie Maya Kaelberer1,2, Sven-Eric Jordt2 1Department of Cellular & Molecular Physiology, Yale University, 2Department of Anesthesiology, Duke University Medical Center Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined. Medicine Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium Takuya Hashimoto1,2,3, Kota Yamamoto1,2,3, Trenton Foster1, Hualong Bai1, Kunihiro Shigematsu4, Alan Dardik1,3 1Department of Surgery and the Vascular Biology and Therapeutics Program, Yale University, 2Department of Vascular Surgery, University of Tokyo, 3Department of Vascular Surgery, VA Connecticut Healthcare Systems, 4Department of Vascular Surgery, International University of Health and Welfare Mita Hospital After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route. Developmental Biology Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres Miguel A. Lopez-Ramirez*1,2, Charles-Félix Calvo*3, Emma Ristori1, Jean-Léon Thomas1,3, Stefania Nicoli1 1Yale Cardiovascular Research Center, Internal Medicine, Yale University, 2Department of Medicine, University of California, San Diego, 3APHP Groupe Hospitalier Pitié-Salpètrière, Université Pierre and Marie Curie Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres. Behavior Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats Robert J. Wickham1, Jinwoo Park2, Eric J. Nunes3, Nii A. Addy1,3,4 1Interdepartmental Neuroscience Program, Yale University, 2Department of Biotechnical and Clinical Laboratory Sciences, School of Medicine and Biomedical Sciences, University at Buffalo, 3Department of Psychiatry, Yale School of Medicine, 4Department of Cellular and Molecular Physiology, Yale School of Medicine Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats. Neuroscience Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy Magalie Bénard1, Alexis Lebon1, Hitoshi Komuro2, David Vaudry1, Ludovic Galas1 1PRIMACEN, Cell Imaging Platform of Normandy, Inserm, IRIB, University of Rouen, 2Department of Neurobiology, School of Medicine, Yale University During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration. Neuroscience Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster Robert W. Fernandez1, Marat Nurilov2, Omar Feliciano2, Ian S. McDonald3, Anne F. Simon3 1Department of Molecular Biophysics and Biochemistry, Yale University, 2Department of Biology, York College/CUNY, 3Department of Biology, Western Ontario University Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster. Engineering Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids Qin Xu1,2, Ivo Peters2, Sam Wilken1,2, Eric Brown3, Heinrich Jaeger1,2 1Department of Physics, The University of Chicago, 2James Franck Institute, The University of Chicago, 3Department of Mechanical Engineering and Materials Science, Yale University Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids. Neuroscience Electrophysiological Recording From Drosophila Labellar Taste Sensilla Rebecca Delventhal1, Aidan Kiely1, John R. Carlson1 1MCDB, Yale University This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila. Behavior Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation Pamela K. Douglas1,2, Maureen Pisani2, Rory Reid1, Austin Head2, Edward Lau2, Ebrahim Mirakhor3, Jennifer Bramen2, Billi Gordon2, Ariana Anderson2, Wesley T. Kerr2, Chajoon Cheong4, Mark S. Cohen1,2 1Neuropsychiatric Institute, University of California, Los Angeles, 2Laboratory of Neuroimaging Technology, University of California, Los Angeles, 3Yale School of Medicine, 4Korean Basic Science Institute We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive ‘cold glove’ instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction. Behavior Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking Evan D. Morris1,2,3,4, Su Jin Kim1,3, Jenna M. Sullivan1,3,4, Shuo Wang3,4, Marc D. Normandin5, Cristian C. Constantinescu6, Kelly P. Cosgrove1,2,3 1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking. Medicine Technical Aspects of the Mouse Aortocaval Fistula Kota Yamamoto1,2, Xin Li1,3, Chang Shu3, Tetsuro Miyata2, Alan Dardik1,4 1The Department of Surgery and the Interdepartmental Program in Vascular Biology and Therapeutics, Yale University, 2Department of Vascular Surgery, The University of Tokyo, 3Department of Vascular Surgery, Central South University, 4VA Connecticut Healthcare Systems The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis. Medicine Mouse Models for Graft Arteriosclerosis Lingfeng Qin1, Luyang Yu2, Wang Min2 1Department of Surgery, Yale University School of Medicine, 2Department of Pathology, Yale University School of Medicine We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA. Neuroscience F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes Silvio Sacchetti1, Kambiz N. Alavian1, Emma Lazrove1, Elizabeth A. Jonas1 1Department 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. 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. Neuroscience Neonatal Subventricular Zone Electroporation David M. Feliciano1, Carlos A. Lafourcade1, Angélique Bordey1 1Department of Neurosurgery and Cellular & Molecular Physiology, Yale University School of Medicine We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells Biology Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization Julie Chaumeil1, Mariann Micsinai1,2,3,4, Jane A. Skok1 1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH). Medicine Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue Elaine Bigelow1,2, Katherine M. Bever1,2, Haiying Xu1,2,3, Allison Yager1, Annie Wu1,2,4, Janis Taube3,5, Lieping Chen6, Elizabeth M. Jaffee1,2,5,7,8, Robert A. Anders1,2,5,8, Lei Zheng1,2,4,5,7 1The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Johns Hopkins University School of Medicine, 3Department of Dermatology, The Johns Hopkins University School of Medicine, 4Department of Surgery, Johns Hopkins University School of Medicine, 5The Sol Goldman Pancreatic Cancer Center, The Johns Hopkins University School of Medicine, 6Yale Cancer Center, Yale School of Medicine, 7The Skip Viragh Center for Pancreatic Cancer, The Johns Hopkins University School of Medicine, 8Department of Pathology, The Johns Hopkins University School of Medicine B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor’s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here. Neuroscience Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices Marko Popovic1, Xin Gao1, Dejan Zecevic1 1Department of Cellular and Molecular Physiology, Yale University School of Medicine An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. Neuroscience Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP Sonia G. Parra*1, Sam S. Vesuna*1, Teresa A. Murray1,2, Michael J. Levene1 1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, Louisiana Tech University Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP. Neuroscience Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods Ifat Levy1,2, Lior Rosenberg Belmaker1, Kirk Manson1, Agnieszka Tymula3, Paul W. Glimcher3,4,5 1Section of Comparative Medicine, Yale School of Medicine, 2Department of Neurobiology, Yale School of Medicine, 3Center for Neural Science, New York University, 4Department of Psychology, New York University, 5Department of Economics, New York University Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain. Neuroscience Preparation of Acute Subventricular Zone Slices for Calcium Imaging Benjamin Lacar1, Stephanie Z. Young1, Jean-Claude Platel1, Angélique Bordey1 1Department of Neurosurgery and Cellular & Molecular Physiology, Yale University School of Medicine A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion. Immunology and Infection Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood Feng Qian1, Ruth R. Montgomery1 1Department of Internal Medicine, Yale University School of Medicine We describe use of ImageStream technology (www.amnis.com), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts. Biology Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP Kun-Yong Kim1, Eriona Hysolli1, In-Hyun Park1 1Yale Stem Cell Center, Department of Genetics, Yale School of Medicine A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed. Neuroscience Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition Gloria Wong1,2, Sanda Dolcos1,3, Ekaterina Denkova1, Rajendra Morey4,5,6, Lihong Wang4,5, Gregory McCarthy6,7, Florin Dolcos1,2,3,8,9 1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University, 5Department of Psychiatry and Behavioral Sciences, Duke University, 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants. Medicine Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety Michelle Hampson1, Teodora Stoica1, John Saksa2, Dustin Scheinost1, Maolin Qiu1, Jitendra Bhawnani1, Christopher Pittenger2,3,4, Xenophon Papademetris1, Todd Constable1 1Department of Diagnostic Radiology, Yale University School of Medicine, 2Department of Psychiatry, Yale University School of Medicine, 3Yale Child Study Center, Yale University School of Medicine, 4Interdepartmental Neuroscience Program, Yale University School of Medicine Here we present a method for training people to control a brain area involved in contamination anxiety and for probing the relationship between contamination anxiety and brain connectivity patterns. Medicine Skeletal Muscle Gender Dimorphism from Proteomics Kalina Dimova1, Lauren Ann Metskas2, Mohini Kulp3, Stylianos P. Scordilis4 1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues. Immunology and Infection Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging Joel R. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis. Bioengineering Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor Angela H. Huang1, Laura E. Niklason1,2 1Department of Biomedical Engineering, Yale University, 2Department of Anesthesiology, Yale University School of Medicine Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts. Neuroscience In vivo Laser Axotomy in C. elegans Alexandra B. Byrne*1, Tyson J. Edwards*1, Marc Hammarlund1 1Department of Genetics, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine 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. Neuroscience Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation Onkar S. Dhande1,2, Michael C. Crair1 1Department of Neurobiology, Yale University, 2Program in Developmental Biology, Baylor College of Medicine We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies. Biology Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method Xueqi Liu1, Hong-Wei Wang1 1Molecular Biophysics and Biochemistry, Yale University This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT). Bioengineering Procedure for Lung Engineering Elizabeth A. Calle*1, Thomas H. Petersen*2, Laura E. Niklason1,3 1Department of Biomedical Engineering, Yale University, 2Department of Biomedical Engineering, School of Medicine, Duke University, 3Department of Anesthesia, Yale University We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time. Biology Protein Crystallization for X-ray Crystallography Moshe A. Dessau1, Yorgo Modis1 1Molecular Biochemistry and Biophysics, Yale University The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography. Biology In vivo Imaging of Deep Cortical Layers using a Microprism Thomas H. Chia1, Michael J. Levene1 1Department of Biomedical Engineering, Yale University Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 μm) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms. Biology Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy Christina Joselevitch1, David Zenisek1 1Cellular and Molecular Physiology, Yale University School of Medicine In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy. Biology Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos. Shiaulou Yuan1, Zhaoxia Sun1 1Department of Genetics, Yale University School of Medicine Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish. Biology Fabrication of Amperometric Electrodes Carolyn M. Pike1, Chad P. Grabner2, Amy B. Harkins1 1Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 2Yale University School of Medicine This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry. Biology Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization Tim Brend1, Scott A. Holley1 1Department 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. Biology In situ Protocol for Butterfly Pupal Wings Using Riboprobes Diane Ramos1, Antonia Monteiro2 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. Biology Proboscis Extension Response (PER) Assay in Drosophila Takashi Shiraiwa1, John R. Carlson1 1Department of Molecular, Cellular, and Developmental Biology, Yale University Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.