Stony Brook University View Institution's Website 25 articles published in JoVE Neuroscience Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation Andre Galenchik-Chan*1, Diane Chernoff*1, Wei Zhou2, Timothy Q. Duong3, Eric R. Muir4 1Renaissance School of Medicine at Stony Brook University, 2Department of Radiology, University of Colorado School of Medicine, 3Department of Radiology, Montefiore-Einstein, 4Department of Radiology, School of Medicine, Stony Brook University We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye diseases. Cancer Research Establishment and Culture of Patient-Derived Breast Organoids Disha Aggarwal1,2, Suzanne Russo1, Payal Naik1, Sonam Bhatia1, David L. Spector1,2 1Cold Spring Harbor Laboratory, Cold Spring Harbor, 2Genetics Graduate Program, Stony Brook University A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids. Cancer Research Longitudinal Intravital Imaging Through Clear Silicone Windows Laura Maiorino*1,2,3, Margaret Shevik*1,4,5, José M. Adrover1, Xiao Han1,6, Elias Georgas7,8, John Erby Wilkinson9, Harrison Seidner1, Leonie Foerschner1, David A. Tuveson1, Yi-Xian Qin8, Mikala Egeblad1 1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals. Bioengineering Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells Michael Tyler Guinn1,2,3, Damiano Coraci*2, Lesia Guinn*2, Gábor Balázsi1,2 1Biomedical Engineering Department, Stony Brook University, 2Laufer Center for Physical and Quantitative Biology, Stony Brook University, 3Stony Brook Medical Scientist Training Program Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study. Immunology and Infection Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes David C. Montrose1,2, Erin M. McNally3, Erika Sue3, Andrew J. Dannenberg3 1Department of Pathology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, 2Stony Brook Cancer Center, Stony Brook, NY, 3Department of Medicine, Weill Cornell Medicine, New York, NY Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided. Medicine A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies Robert A. Honkanen1, Liqun Huang2,3, Basil Rigas2 1Department of Ophthalmology, Stony Brook University, 2Department of Preventive Medicine, Stony Brook University, 3Medicon Pharmaceuticals, Inc. This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents. Medicine Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits Robert A. Honkanen1, Liqun Huang2,3, Wei Huang1, Basil Rigas2,4 1Department of Ophthalmology, Stony Brook University, 2Department of Medicine, Stony Brook University, 3Medicon Pharmaceuticals, Inc, 4Department of Preventive Medicine, Stony Brook University A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics. 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. Behavior 3D Kinematic Gait Analysis for Preclinical Studies in Rodents Jeffrey Wong1, Prithvi K. Shah1,2 1Department of Neurobiology and Behavior, Stony Brook University, 2Department of Physical Therapy, Stony Brook University Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies. Environment Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction Cecilia S.N. Cheung1,2, Donald J. Weidner1, Li Li1, Philip G. Meredith3, Haiyan Chen1, Matthew Whitaker1, Xianyin Chen4 1Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook University We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials. Immunology and Infection Isolating Lymphocytes from the Mouse Small Intestinal Immune System Zhijuan Qiu1, Brian S. Sheridan1 1Center for Infectious Diseases, Department of Molecular Genetics and Microbiology, Stony Brook University Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system. Behavior Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation Erin V.L. Vasudevan1,2, Rami J. Hamzey1,2, Eileen M. Kirk2 1Physical Therapy, School of Health Technology and Management, Stony Brook University, 2Motor Learning Lab, Moss Rehabilitation Research Institute, Einstein Healthcare Network We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments). Developmental Biology Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis Patricia Bossert1, Gerald H. Thomsen1 1Department of Biochemistry and Cell Biology, Center for Developmental Genetics, Stony Brook University Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures. Biochemistry Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay Jonathan Fischer1, Roger A. Johnson2, Elizabeth Boon1 1Department of Chemistry, Stony Brook University, 2Department of Physiology and Biophysics, Stony Brook University We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins. Chemistry Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives Pengju Feng1,2, Ming-Yu Ngai1,2 1Department of Chemistry, State University of New York at Stony Brook, 2Institute of Chemical Biology and Drug Discovery, State University of New York at Stony Brook An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported. Neuroscience Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons Chongbo Zhong1, David A. Talmage2, Lorna W. Role1 1Department of Neurobiology and Behavior, Stony Brook University, 2Department of Pharmacological Science, Stony Brook University We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity. Immunology and Infection Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans Arielle M. Bryan*1, Amir M. Farnoud*1, Visesato Mor1, Maurizio Del Poeta1 1Department of Molecular Genetics and Microbiology, Stony Brook University In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections. Immunology and Infection Culturing Microglia from the Neonatal and Adult Central Nervous System Robert Bronstein*1,2, Luisa Torres*2,3, Jillian C. Nissen*2,3, Stella E. Tsirka2 1Program in Neuroscience, Stony Brook University, 2Department of Pharmacological Sciences, Stony Brook University, 3Program in Molecular and Cellular Pharmacology, Stony Brook University We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection. Biology A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types Haipeng Xing1, Willey Liao1,2, Yifan Mo1,2, Michael Q. Zhang2,3 1Department of Applied Mathematics & Statistics, Stony Brook University, 2Computational Biology and Bioinformatics, Cold Spring Harbor Laboratory, 3Department of Molecular and Cell Biology, University of Texas at Dallas Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment. Immunology and Infection Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry Frederick A. Rubino1, Yoon Hyeun Oum1, Lakshmi Rajaram1, Yanjie Chu1, Isaac S. Carrico1 1Department of Chemistry, Stony Brook University Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification. Biology Measuring Fast Calcium Fluxes in Cardiomyocytes Urszula Golebiewska1, Suzanne Scarlata2 1Department of Biological Sciences and Geology, Queensborough Community College, 2Department of Physiology & Biophysics, Stony Brook University We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes. Immunology and Infection Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines Joyce Hui-Yuen1,2, Shane McAllister1,2, Siva Koganti2, Erik Hill2, Sumita Bhaduri-McIntosh1,2,3,4 1Stony Brook Children's Hospital, State University of New York at Stony Brook, 2Department of Pediatrics, State University of New York at Stony Brook, 3Department of Molecular Genetics, State University of New York at Stony Brook, 4Department of Microbiology, State University of New York at Stony Brook We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection. Neuroscience Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues Ryan W. O'Meara1,2, Scott D. Ryan1, Holly Colognato3, Rashmi Kothary1,2,4 1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs). Neuroscience Studying the Integration of Adult-born Neurons Yan Gu1, Stephen Janoschka1, Shaoyu Ge1 1Department of Neurobiology & Behavior, State University of New York at Stony Brook A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration. Biology A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment Shoko Ueki1, Benjamin L. Meyers1, Farzana Yasmin2, Vitaly Citovsky2 1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
