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In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells Timothy J Petros1, Alexandra Rebsam1, Carol A Mason2, 1 Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties. Related Products: |
In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos William Walantus, Laura Elias, Arnold Kriegstein In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video. Related Products: |
In Ovo Electroporations of HH Stage 10 Chicken Embryos Marissa C. Blank1, Victor Chizhikov2, Kathleen J. Millen2 Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed. Related Products: |
Warner Instruments
Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs Hui Zhang1, 2, Niko G. Gubernator3, 4, Minerva Yue1, Roland G. W. Staal1, Eugene V. Mosharov1, Daniela Pereira1, Vojtech Balsanek3, Paul A. Vadola3, Bipasha Mukherjee5, Robert H. Edwards5, David Sulzer1, 2, 6, Dalibor Sames3 A new means to measure neurotransmission optically using fluorescent dopamine analogs. Related Products: |
Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes Eiji Shigetomi, Baljit S. Khakh We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy. Related Products: |
Calcium Imaging of Cortical Neurons using Fura-2 AM Odmara L Barreto-Chang, Ricardo E Dolmetsch Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades. Related Products: |
Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber Javeed Shaikh Mohammed1, Hugo Caicedo1, Christopher P. Fall2, David T. Eddington1 We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters. Related Products: |
Medical Systems Research
Zebrafish Brain Ventricle Injection Jennifer H. Gutzman1, Hazel Sive1, 2 After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo. Related Products: |
Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy Ellie Graeden1, 2, Hazel Sive1, 2 In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain. Related Products: |
Microinjection of Zebrafish Embryos to Analyze Gene Function Jonathan N. Rosen1, 2, Michael F. Sweeney1, John D. Mably1, 2 This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development. Related Products: |
Harvard Apparatus
Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium Georg Wiese1, Steven R. Barthel2, Charles J. Dimitroff2 This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces. Related Products: |
Implantation of Engineered Tissue in the Rat Heart Bjoern Sill, Ivan V. Alpatov, Christina A. Pacak, Douglas B. Cowan Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat. Related Products: |
Visualizing Single-molecule DNA Replication with Fluorescence Microscopy Nathan A. Tanner, Joseph J. Loparo, Antoine M. van Oijen This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time. Related Products: |
Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers Justin R. Seymour, Marcos, Roman Stocker The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described. Related Products: |
Method for Measurement of Viral Fusion Kinetics at the Single Particle Level Daniel L. Floyd1, Stephen C. Harrison1, 2, Antoine M. van Oijen1 We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation. Related Products: |
Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells William N. White1, Palaniappan Sethu2 An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocytelysis that ensures isolation of high quality sample without cell loss. Related Products: |
