Oregon Health and Science University View Institution's Website 39 articles published in JoVE Neuroscience Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina Ryan M. Hecht1, Qing Shi1, Tavita R. Garrett2, Benjamin Sivyer2, Catherine Morgans1 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. Medicine Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy Zhiqiang Hou1, Yunpei Zhang1, Lingling Neng1, Jinhui Zhang, Xiaorui Shi1 1Oregon Hearing Research Center, Department of Otolaryngology/Head & Neck Surgery, Oregon Health & Science University An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions. Neuroscience Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy Suvithanandhini Loganathan*1, Hannah E. Ball*1, Ernesto Manzo1,2, Daniela C. Zarnescu1,3 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. 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. Mall1,2,5,6,7, Carsten Schultz1,3,8 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. Medicine Optical Clearing and Imaging of Immunolabeled Kidney Tissue Turgay Saritas1, Victor G. Puelles1,2,3, Xiao-Tong Su4, David H. Ellison4,5,6, Rafael Kramann1,7 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. Neuroscience Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy Bart C. Jongbloets*1, Lei Ma*1, Tianyi Mao1, Haining Zhong1 1Vollum Institute, Oregon Health & Science University A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKARα, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion. Cancer Research Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer Rebecca Smith1, Kaylyn Devlin1, David Kilburn1, Sean Gross1, Damir Sudar2, Elmar Bucher1, Michel Nederlof2, Mark Dane1, Joe W. Gray1, Laura Heiser1, James E. Korkola1 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. Neuroscience Localization of the Locus Coeruleus in the Mouse Brain Katharina Schmidt1, Bilal Bari2, Martina Ralle3, Clorissa Washington-Hughes1, Abigael Muchenditsi1, Evan Maxey4, Svetlana Lutsenko1 1Department of Physiology, Johns Hopkins University, School of Medicine, 2Department of Neuroscience, Johns Hopkins University, 3Department of Molecular and Medical Genetics, OHSU, 4X-ray science division, Advanced Photon Source, Argonne National Laboratory The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus. Neuroscience In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films Skyler L. Jackman1, Christopher H. Chen2, Wade G. Regehr2 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. Biology Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors Maria M. Steele1, Madeline J. Churchill2, Alec P. Breazeale1, Ryan S. Lane1, Nicholas A. Nelson1, Amanda W. Lund1,2,3,4 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. Biology Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy Katsuyuki Matsushita1, Kirsti Golgotiu1, Daniel J. Orton2, Richard D. Smith2, Karin D. Rodland2, Paul D. Piehowski2, Michael P. Hutchens1,3 1Anesthesiology & Perioperative Medicine, Oregon Health & Science University, 2Environmental and Biological Services Division, Pacific Northwest National Laboratory, 3Operative Care Division, Portland Veterans Affairs Medical Center We present use of 2-photon microscopy to place a micropipette within Bowman's urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies. Bioengineering Light-sheet Fluorescence Microscopy for the Study of the Murine Heart Yichen Ding1, Zachary Bailey2, Victoria Messerschmidt2, Jun Nie3, Richard Bryant2, Sandra Rugonyi4, Peng Fei3, Juhyun Lee1,2, Tzung K. Hsiai1 1Department of Bioengineering, University of California Los Angeles, 2Department of Bioengineering, University of Texas at Arlington, 3School of Optical and Electronic Information, Huazhong University of Science and Technology, 4Department of Biomedical Engineering, OSHU This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart. Behavior Methodology for Establishing a Community-Wide Life Laboratory for Capturing Unobtrusive and Continuous Remote Activity and Health Data Jeffrey Kaye1, Christina Reynolds1, Molly Bowman1, Nicole Sharma1, Thomas Riley1, Ona Golonka1, Jonathan Lee1, Charlie Quinn1, Zachary Beattie1, Johanna Austin1, Adriana Seelye1, Katherine Wild1, Nora Mattek1 1Department of Neurology, ORCATECH - Oregon Center for Aging & Technology, Oregon Health & Science University Unobtrusive sensors and pervasive computing technology incorporated into the daily home life of older adults enables meaningful health and activity changes to be recorded continuously for months to years, providing ecologically valid, high frequency, multi-domain data for research or clinical use. Biology A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins John M. Powers1, Xiao Lan Chang1, Zhen Song1, Hiroyuki Nakai1,2 1Department of Molecular and Medical Genetics, Oregon Health & Science University, 2Department of Molecular Microbiology and Immunology, Oregon Health & Science University This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes. 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 Maloyan1 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. Biology An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus Shane C. McAllister1, Ryan S. Hanson1, Kyleen N. Grissom1, Sara Botto2, Ashlee V. Moses2 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. Cancer Research Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis Charles E. Gast1, Aubie K. Shaw1,2, Melissa H. Wong1,3, Lisa M. Coussens1,3 1Cell, Developmental & Cancer Biology, Oregon Health & Science University, 2University of Minnesota, 3Knight Cancer Institute, Oregon Health & Science University Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases. Biology Visualizing the Early Stages of Phagocytosis Ali Rashidfarrokhi*2, Veronica Richina*2, Fikadu G. Tafesse1,2 1Department of Molecular Microbiology & Immunology, Oregon Health & Science University, 2Ragon Institute of MGH, MIT and Harvard Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads. Cancer Research A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer Erica T. Goddard1, Jacob Fischer1, Pepper Schedin1 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. Neuroscience Mass Histology to Quantify Neurodegeneration in Drosophila Elizabeth R. Sunderhaus1, Doris Kretzschmar1 1Oregon Institute of Occupational Health Sciences, Oregon Health & Sciences University Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample. Biochemistry Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram Jonathan A. Coleman1, Evan M. Green2, Eric Gouaux1,3 1Vollum Institute, Oregon Health & Science University, 2Graduate Group in Biophysics, University of California, San Francisco, 3Howard Hughes Medical Institute, Oregon Health & Science University This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels. Bioengineering Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) Andrew Nickerson1,2,3, Tao Huang1,2,3, Li-Jung Lin1,2,3, Xioalin Nan1,2,3 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. Biology Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle William C.W. Chen1, Arman Saparov2,3, Mirko Corselli4, Mihaela Crisan5, Bo Zheng6, Bruno Péault7,8, Johnny Huard9 1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7 Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia. Neuroscience Super-resolution Imaging of Neuronal Dense-core Vesicles Bethe A. Scalettar1,2, Daniel Shaver2, Stefanie Kaech3, Janis E. Lochner2,4 1Department of Physics, Lewis & Clark College, 2Program in Biochemistry and Molecular Biology, Lewis & Clark College, 3Jungers Center for Neuroscience Research, Oregon Health & Science University, 4Department of Chemistry, Lewis & Clark College We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image. Bioengineering Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy Esra Güç*1, Manuel Fankhauser*1, Amanda W. Lund1,2, Melody A. Swartz1, Witold W. Kilarski1 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. Bioengineering Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope Kevin G. Phillips1,2, Sandra M. Baker-Groberg1, Owen J.T. McCarty1,3 1Department of Biomedical Engineering, Oregon Health & Science University, School of Medicine, 2Department of Dermatology, Oregon Health & Science University, School of Medicine, 3Department of Cell & Developmental Biology, Division of Hematology & Medical Oncology, Knight Cancer Institute, Oregon Health & Science University, School of Medicine We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery. Neuroscience Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways Stacy L. Fairbanks*1, Rebekah Vest*1, Saurabh Verma2, Richard J. Traystman1,3, Paco S. Herson1,3 1Department of Anesthesiology, University of Colorado School of Medicine, 2Oregon National Primate Research Center, Oregon Health & Science University, 3Department of Pharmacology, University of Colorado School of Medicine Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling. Neuroscience Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons Robert P. Irwin1, Charles N. Allen1,2 1Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, 2Department of Behavioral Neuroscience, Oregon Health & Science University Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration. Immunology and Infection Tractable Mammalian Cell Infections with Protozoan-primed Bacteria Samuel L. Drennan1, Amrita Lama1, Ben Doron1, Eric D. Cambronne1 1Department of Molecular Microbiology & Immunology, Oregon Health & Science University This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types. Medicine A Simple Method of Mouse Lung Intubation Sandhya Das1, Kelvin MacDonald2, Herng-Yu Sucie Chang1, Wayne Mitzner1 1Department of Environmental Health Sciences, Program in Respiratory Biology and Lung Disease, Johns Hopkins Bloomberg School of Public Health, 2Department of Pediatrics, Oregon Health Sciences University This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea. Biology Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays Katharina L. Dürr1,2, Neslihan N. Tavraz1, Susan Spiller1, Thomas Friedrich1 1Institute of Chemistry, Technical University of Berlin, 2The Vollum Institute, Oregon Health & Science University We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies. Biology Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization Leslie Smith1, Mathew Thayer1 1Department of Biochemistry and Molecular Biology, Knight Cancer Institute, Oregon Health & Science University A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell. Medicine Doppler Optical Coherence Tomography of Retinal Circulation Ou Tan1, Yimin Wang1, Ranjith K. Konduru2, Xinbo Zhang1, SriniVas R. Sadda2, David Huang1 1Department of Ophthalmology, Oregon Health and Science University, 2Department of Ophthalmology, University of Southern California Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software. Neuroscience Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation Lingyan Wang1, Han Jiang1, John V. Brigande1 1Oregon Hearing Research Center, Oregon Health & Science University The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques. Neuroscience Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices Mean-Hwan Kim1, Evan Vickers1, Henrique von Gersdorff1 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. Medicine Method to Measure Tone of Axial and Proximal Muscle Victor S. Gurfinkel1, Timothy W. Cacciatore2, Paul J. Cordo1, Fay B. Horak3 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. Medicine Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury Michael P. Hutchens1, Richard J. Traystman2, Tetsuhiro Fujiyoshi1, Shin Nakayama1, Paco S. Herson1 1Department of Anesthesiology and Perioperative Medicine, Oregon Health & Sciences University, 2Department of Pharmacology, University of Colorado Denver A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI. Neuroscience Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish Hua Wen1, Paul Brehm1 1Vollum Institute, Oregon Health and Sciences University Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Neuroscience DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices Gail K. Seabold1, James B. Daunais2, Andrew Rau3, Kathleen A. Grant3, Veronica A. Alvarez1 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.