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Columbia University College of Physicians and Surgeons

12 ARTICLES PUBLISHED IN JoVE

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Biology

Drosophila Larval NMJ Dissection
Jonathan R. Brent 1, Kristen M. Werner 1, Brian D. McCabe 1
1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

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Biology

Drosophila Larval NMJ Immunohistochemistry
Jonathan Brent 1, Kristen Werner 1, Brian D. McCabe 1
1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

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Biology

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae
Wendy Imlach 1, Brian D. McCabe 1
1Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

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Biology

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells
Timothy J Petros 1, Alexandra Rebsam 1, Carol A Mason 1,2
1Departments of Pathology and Cell Biology, and Neuroscience, Columbia University College of Physicians and Surgeons, 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons

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.

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Immunology and Infection

Recombinant Retroviral Production and Infection of B Cells
Celia Keim 1, Veronika Grinstein 1, Uttiya Basu 1,2
1Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, 2Herbert Irving Comprehensive Cancer Center, Columbia University

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

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Biology

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
Jessica M. Skeie 1,2, Stephen H. Tsang 3, Vinit B. Mahajan 1,2
1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

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Medicine

Mouse Eye Enucleation for Remote High-throughput Phenotyping
Vinit B. Mahajan 1,2, Jessica M. Skeie 1,2, Amir H. Assefnia 2,3, MaryAnn Mahajan 1,2, Stephen H. Tsang 2,4
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

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Medicine

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Katherine J. Wert 1,2, Jessica M. Skeie 3,4, Richard J. Davis 1, Stephen H. Tsang 1,3, Vinit B. Mahajan 3,4
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University , 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University , 3Omics Laboratory, University of Iowa , 4Department of Ophthalmology and Visual Sciences, University of Iowa

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

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Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma
Michael Li 1,2, Ravi R. Pathak 5, Esther Lopez-Rivera 3, Scott L. Friedman 1, Julio A. Aguirre-Ghiso 4, Andrew G. Sikora 5
1Department of Medicine, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, 2Department of Otolaryngology, Icahn School of Medicine at Mount Sinai, 3Division of Nephrology, Columbia University College of Physicians and Surgeons, 4Departments of Medicine, Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, 5Bobby R. Alford Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

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Biochemistry

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis
Thiago Cabral *1,2,7,8, Marcus A. Toral *3,4, Gabriel Velez 3,4, James E. DiCarlo 1,2, Anuradha M. Gore 3, MaryAnn Mahajan 3, Stephen H. Tsang 1,2, Alexander G. Bassuk 5,6, Vinit B. Mahajan 3,9
1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

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Medicine

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia
Mariya Zyablitskaya 1, E. Laura Munteanu 2, Takayuki Nagasaki 1, David C. Paik 1
1Department of Ophthalmology, Columbia University College of Physicians and Surgeons, 2Confocal and Specialized Microscopy Shared Resource, Herbert Irving Comprehensive Cancer Center, Columbia University

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using second harmonic generation imaging and differential scanning calorimetry.

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Neuroscience

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue
Jiwon Woo 1,2,3, Eunice Yoojin Lee 4, Hyo-Suk Park 1,3, Jeong Yoon Park 1,3, Yong Eun Cho 1,2,3
1Department of Neurosurgery, Gangnam Severance Hospital, Yonsei University College of Medicine, 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University, 3The Spine and Spinal Cord Institute, Biomedical Center, Gangnam Severance Hospital, Yonsei University College of Medicine, 4Columbia University College of Physicians and Surgeons

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

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