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University of Tübingen

21 ARTICLES PUBLISHED IN JoVE

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Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution
Yao Zhang *1,2, Petra Füger *1, Shabab B. Hannan 1,2, Jeannine V. Kern 1, Bronwen Lasky 1, Tobias M. Rasse 1
1Junior Research Group Synaptic Plasticity, Hertie Institute for Clinical Brain Research, University of Tübingen, 2Graduate School of Cellular and Molecular Neuroscience, University of Tübingen

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

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Biology

Preparation of Quality Inositol Pyrophosphates
Omar Loss 1, Cristina Azevedo 1, Zsolt Szijgyarto 1, Daniel Bosch 1, Adolfo Saiardi 1
1Medical Research Council (MRC), Cell Biology Unit and Laboratory for Molecular Cell Biology, University College London

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

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Medicine

High Content Screening in Neurodegenerative Diseases
Shushant Jain 1, Ronald E. van Kesteren 2, Peter Heutink 1
1Department of Clinical Genetics, VU University Medical Center, 2Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

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Medicine

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease
Lena F. Burbulla 1,2, Rejko Krüger 1,2
1German Center for Neurodegenerative Diseases, DZNE, 2Laboratory of Functional Neurogenomics, Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

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Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain
Carola Huber 1, A. Alwin Prem Anand 1, Manfred Mauz 1, Peter Künstle 1, Wolfgang Hupp 1, Bernhard Hirt 1, Andrea Wizenmann 1
1Department of Experimental Embryology, Institute of Anatomy, University of Tübingen

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

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Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo
Christopher F. Schuster 1, Ralph Bertram 1
1Department of Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), Faculty of Science, University of Tübingen

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

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Neuroscience

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina
Manoj Kulkarni 1,2, Timm Schubert 1,3, Tom Baden 1,2,3, Bernd Wissinger 4,5, Thomas Euler 1,2,3, Francois Paquet-Durand 1
1Institute for Ophthalmic Research, University of Tübingen, 2Graduate School of Cellular & Molecular Neuroscience, University of Tübingen, 3Bernstein Centre for Computational Neuroscience, University of Tübingen, 4Molecular Genetics Laboratory, University of Tübingen, 5Centre for Ophthalmology, University of Tübingen

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

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Neuroscience

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices
Daniel Bosch 1, Douglas Asede 2, Ingrid Ehrlich 1
1Hertie Institute for Clinical Brain Research and Werner Reichardt Centre for Integrative Neuroscience, University of Tuebingen, 2Max Planck Florida Institute for Neuroscience

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

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Neuroscience

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala
Sabine Schönherr 1, Anna Seewald 1, Yu Kasugai 1, Daniel Bosch 2, Ingrid Ehrlich 2, Francesco Ferraguti 1
1Department of Pharmacology, Medical University of Innsbruck, 2Hertie Institute for Clinical Brain Research and Centre for Integrative Neuroscience, University of Tübingen

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

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Chemistry

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique
Mareike Binder 1, Claudio Schrenk 1, Andreas Schnepf 1
1Chemistry Department, Institute of Inorganic Chemistry, University of Tübingen

The disproportionation reaction of a metastable Sn(I) chloride solution, obtained via the preparative co-condensation technique, is used for the synthesis of a metalloid tin cluster compound.

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Engineering

3D Printing of Biomolecular Models for Research and Pedagogy
Eduardo Da Veiga Beltrame 1, James Tyrwhitt-Drake 2, Ian Roy 3, Raed Shalaby 4, Jakob Suckale 4, Daniel Pomeranz Krummel 5
1Department of Physics, Brandeis University, 2Bioinformatics and Computational Biosciences Branch (BCBB), NIH/NIAID/OD/OSMO/OCICB, 3Library/LTS/MakerLab, Brandeis University, 4Interfaculty Institute of Biochemistry (IFIB), University of Tübingen, 5Winship Cancer Institute, Emory University School of Medicine

Physical models of biomolecules can facilitate an understanding of their structure-function for the researcher, aid in communication between researchers, and serve as an educational tool in pedagogical endeavors. Here, we provide detailed guidance for the 3D printing of accurate models of biomolecules using fused filament fabrication desktop 3D printers.

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Cancer Research

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia
Alexander R. Fuchs 1, Melanie Märklin 1, Jonas S. Heitmann 1, Stefan Futterknecht 1, Michael Haap 2, Stefan Wirths 1, Hans-Georg Kopp 1, Clemens Hinterleitner 1, Daniela Dörfel 1, Martin R. Müller 1
1Dept. of Hematology, Oncology and Immunology, University of Tübingen, 2Dept. of Endocrinology, Diabetology, Clinical Pathology and Metabolism, University of Tübingen

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

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Immunology and Infection

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses
Anna Lange 1, Andrea Schäfer 1, Julia-Stefanie Frick 1
1Department for Medical Microbiology, Hygiene, Interfacultary Institute for Microbiology, Infection Medicine, University of Tübingen

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

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Neuroscience

Use of Real-Time Functional Magnetic Resonance Imaging-Based Neurofeedback to Downregulate Insular Cortex in Nicotine-Addicted Smokers
Mohit Rana 1,2, Sergio Ruiz 1,2, Andrea Sánchez Corzo 1,2, Axel Muehleck 4, Sandra Eck 4, César Salinas 5, Francisco Zamorano 5,6, Claudio Silva 5, Massimiliano Rea 4, Anil Batra 4, Niels Birbaumer 1,7,8, Ranganatha Sitaram 1,2,9
1Departamento de Psiquiatría, Escuela de Medicina, Centro Interdisciplinario de Neurociencias, Pontificia Universidad Católica de Chile, 2Laboratory for Brain-Machine Interfaces and Neuromodulation, Pontificia Universidad Católica de Chile, 3Institute of Medical Psychology and Behavioral Neurobiology, University of Tübingen, 4Department of Psychiatry and Psychotherapy, University of Tübingen, 5Unidad de Imágenes Cuantitativas Avanzadas, Departamento de Imaágenes, Facultad de Medicina Clínica Alemana, Universidad del Desarrollo, 6División de Neurociencia, Centro de Investigación en Complejidad Social (neuroCICS), Facultad de Gobierno, Universidad del Desarrollo, 7Institute di Ricovero e Cura a Carattere Scientifico, 8Wyss Center for Bio and Neuroengineering, 9Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile

In real-time functional magnetic resonance imaging (rtfMRI), brain activity is experimentally manipulated as an independent variable, and behavior is measured as a dependent variable. The protocol presented here focuses on the practical use of rtfMRI as a therapeutic tool for psychiatric disorders such as nicotine addiction.

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Behavior

Brain State-dependent Brain Stimulation with Real-time Electroencephalography-Triggered Transcranial Magnetic Stimulation
Maria-Ioanna Stefanou 1,2, David Baur 1,2, Paolo Belardinelli 1,2, Til Ole Bergmann 1,2, Corinna Blum 1,2, Pedro Caldana Gordon 1,2, Jaakko O. Nieminen 1,2,3, Brigitte Zrenner 1,2, Ulf Ziemann 1,2, Christoph Zrenner 1,2
1Department of Neurology & Stroke, University of Tübingen, 2Hertie Institute for Clinical Brain Research, University of Tübingen, 3Department of Neuroscience and Biomedical Engineering, Aalto University

This paper describes real-time electroencephalography-triggered transcranial magnetic stimulation to study and modulate human brain networks.

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Education

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency
Marianne Theile 1, Linus Wiora 1, Dominik Russ 1, Jonas Reuter 1, Hiroshi Ishikawa 2, Christian Schwerk 3, Horst Schroten 3, Stefan Mogk 1
1Interfaculty Institute of Biochemistry, University of Tübingen, 2Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 3Department of Pediatrics, Medical Faculty Mannheim, Heidelberg University

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

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Neuroscience

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder
Rui Wang 1, Zi-tong Huang 3, Wen-kang Ren 1, Jiao Zhang 1, Yao Zhang 1, Bo Tan 3, Ping Huang 1,2, Hong-ying Cao 1,2
1School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, 2Dongguan & Guangzhou University of Chinese Medicine Cooperative Academy of Mathematical Engineering for Chinese Medicine, Guangzhou University of Chinese Medicine, 3School of Basic Medical Sciences, Guangzhou University of Chinese Medicine

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

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Biology

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring
Ashutosh Dhingra *1, Joachim Täger *1, Elisangela Bressan *1, Salvador Rodriguez-Nieto 1, Manmeet-Sakshi Bedi 1, Stefanie Bröer 1, Eldem Sadikoglou 1, Noémia Fernandes 2, Melissa Castillo-Lizardo 1, Patrizia Rizzu 2, Peter Heutink 1,3
1Genome Biology of Neurodegenerative Diseases, German Center for Neurodegenerative Diseases (DZNE), 2Applied Genomics for Neurodegenerative Diseases, German Center for Neurodegenerative Diseases (DZNE), 3Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

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Neuroscience

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium
Soumaya Belhadj *1, Arianna Tolone *1, Gustav Christensen 1, Soumyaparna Das 1, Yiyi Chen 1, François Paquet-Durand 1
1Institute for Ophthalmic Research, University of Tübingen

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

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Biology

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae
Deeksha Singh 1, Sara Blicharz 1, Karolina Stefanowicz 1, Laura Ragni 2, Kornel Michalak 3, Agnieszka Bagniewska-Zadworna 3, Robert Malinowski 1
1Integrative Plant Biology Team, Institute of Plant Genetics Polish Academy of Sciences, 2ZMBP-Center for Plant Molecular Biology, University of Tübingen, 3Department of General Botany, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

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Immunology and Infection

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test
Julia Häring *1, Tanja Michel *1, Matthias Becker 1, Daniel Junker 1, Tatia Tchitchagua 2, Olaf Leschnik 2, Berit Lange 3,4, Stefanie Castell 3,4, Gérard Krause 3,4, Monika Strengert 3, Alex Dulovic 1, Nicole Schneiderhan-Marra 1
1NMI Natural and Medical Sciences Institute at the University of Tübingen, 2Department of Neurology, Sächsisches Krankenhaus Rodewisch, 3Department of Epidemiology, Helmholtz Centre for Infection Research, 4German Centre for Infection Research (DZIF)

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

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