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Oregon Health and Science University

16 ARTICLES PUBLISHED IN JoVE

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Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices
Gail K. Seabold 1, James B. Daunais 2, Andrew Rau 3, Kathleen A. Grant 3, Veronica A. Alvarez 1
1Section on Neuronal Structure, Laboratory for Integrative Neuroscience, NIAAA, NIH, 2Department Physiology and Pharmacology, Wake Forest University Health Sciences, 3Oregon National Primate Research Center, Division of Neuroscience, Oregon Health and Science University

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

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Neuroscience

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction
Rebecca Smith 1, J. Paul Taylor 1
1Developmental Neurobiology, St. Jude Children’s Research Hospital

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

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Neuroscience

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices
Mean-Hwan Kim 1, Evan Vickers 1, Henrique von Gersdorff 1
1The Vollum Institute, Oregon Health and Science University

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

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Medicine

Method to Measure Tone of Axial and Proximal Muscle
Victor S. Gurfinkel 1, Timothy W. Cacciatore 2, Paul J. Cordo 1, Fay B. Horak 3
1Department of Biomedical Engineering, Oregon Health and Science University, 2UCL Institute of Neurology, Queen Square, 3Department of Neurology, Oregon Health and Science University

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

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Bioengineering

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Esra Güç *1, Manuel Fankhauser *1, Amanda W. Lund 1,2, Melody A. Swartz 1, Witold W. Kilarski 1
1Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne, 2Department of Cell and Developmental Biology and Knight Cancer Institute, Oregon Health & Science University

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

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Bioengineering

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)
Andrew Nickerson 1,2,3, Tao Huang 1,2,3, Li-Jung Lin 1,2,3, Xioalin Nan 1,2,3
1Department of Biomedical Engineering, Oregon Health and Science University, 2Knight Cancer Institute, Oregon Health and Science University, 3OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

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Biology

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus
Shane C. McAllister 1, Ryan S. Hanson 1, Kyleen N. Grissom 1, Sara Botto 2, Ashlee V. Moses 2
1Division of Pediatric Infectious Diseases, University of Minnesota Medical School, 2Vaccine and Gene Therapy Institute, Oregon Health and Science University

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

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Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer
Erica T. Goddard 1, Jacob Fischer 1, Pepper Schedin 1
1Department of Cell, Developmental and Cancer Biology, Oregon Health and Science University

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

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Medicine

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta
Bailey Simon *1, Matthew Bucher *2, Alina Maloyan 1
1Knight Cardiovascular Institute, Oregon Health and Science University, 2Department of Obstetrics and Gynecology, Oregon Health and Science University

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNFα recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

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Biology

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors
Maria M. Steele 1, Madeline J. Churchill 2, Alec P. Breazeale 1, Ryan S. Lane 1, Nicholas A. Nelson 1, Amanda W. Lund 1,2,3,4
1Department of Cell, Developmental, & Cancer Biology, Oregon Health and Science University, 2Department of Molecular Microbiology & Immunology, Oregon Health and Science University, 3Department of Dermatology, Oregon Health and Science University, 4Knight Cancer Institute, Oregon Health and Science University

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from naïve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

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Neuroscience

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films
Skyler L. Jackman 1, Christopher H. Chen 2, Wade G. Regehr 2
1Vollum Institute, Oregon Health and Science University, 2Harvard Medical School

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

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Cancer Research

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer
Rebecca Smith 1, Kaylyn Devlin 1, David Kilburn 1, Sean Gross 1, Damir Sudar 2, Elmar Bucher 1, Michel Nederlof 2, Mark Dane 1, Joe W. Gray 1, Laura Heiser 1, James E. Korkola 1
1Department of Biomedical Engineering, Knight Cancer Institute, Oregon Health and Science University, 2Quantitative Imaging Systems LLC

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

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Medicine

Optical Clearing and Imaging of Immunolabeled Kidney Tissue
Turgay Saritas 1, Victor G. Puelles 1,2,3, Xiao-Tong Su 4, David H. Ellison 4,5,6, Rafael Kramann 1,7
1Division of Nephrology and Clinical Immunology, University Hospital RWTH Aachen, 2III. Department of Medicine, University Medical Center, Hamburg-Eppendorf, 3Department of Nephrology, Monash Health, 4Division of Nephrology and Hypertension, Oregon Health and Science University, 5Renal Section, Veterans Affairs Portland Health Care System, 6Fondation LeDucq Transatlantic Networks of Excellence, 7Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

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Immunology and Infection

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples
Dario L. Frey *1,2, Matteo Guerra *1,2,3,4, Marcus A. Mall 1,2,5,6,7, Carsten Schultz 1,3,8
1Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 2Dept. of Translational Pulmonology, University of Heidelberg, 3Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL), University of Heidelberg, 4Faculty of Biosciences, Collaboration for Joint Ph.D. Degree between EMBL and Heidelberg University, University of Heidelberg, 5Dept. of Pediatric Pulmonology, Immunology and Critical Care Medicine, Charité – Universitätsmedizin Berlin, 6Berlin Institute of Health (BIH), 7German Center for Lung Research (DZL), Associated Partner Site, Berlin, 8Dept. of Chemical Physiology and Biochemistry, Oregon Health and Science University

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

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Neuroscience

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy
Suvithanandhini Loganathan *1, Hannah E. Ball *1, Ernesto Manzo 1,2, Daniela C. Zarnescu 1,3
1Department of Molecular and Cellular Biology, University of Arizona, Tucson, 2Vollum Institute, Oregon Health and Science University, 3Department of Neuroscience, University of Arizona, Tucson

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

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Neuroscience

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina
Ryan M. Hecht 1, Qing Shi 1, Tavita R. Garrett 2, Benjamin Sivyer 2, Catherine Morgans 1
1Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, 2Casey Eye Institute, Oregon Health and Science University

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.

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