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The Johns Hopkins University School of Medicine

16 ARTICLES PUBLISHED IN JoVE

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Medicine

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
Elaine Bigelow 1,2, Katherine M. Bever 1,2, Haiying Xu 1,2,3, Allison Yager 1, Annie Wu 1,2,4, Janis Taube 3,5, Lieping Chen 6, Elizabeth M. Jaffee 1,2,5,7,8, Robert A. Anders 1,2,5,8, Lei Zheng 1,2,4,5,7
1The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Johns Hopkins University School of Medicine, 3Department of Dermatology, The Johns Hopkins University School of Medicine, 4Department of Surgery, Johns Hopkins University School of Medicine, 5The Sol Goldman Pancreatic Cancer Center, The Johns Hopkins University School of Medicine, 6Yale Cancer Center, Yale School of Medicine, 7The Skip Viragh Center for Pancreatic Cancer, The Johns Hopkins University School of Medicine, 8Department of Pathology, The Johns Hopkins University School of Medicine

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor’s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

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Clinical Skills

Basic Life Support: Cardiopulmonary Resuscitation and Defibrillation
Julianna Jung 1
1The Johns Hopkins University School of Medicine

Basic Life Support: Cardiopulmonary Resuscitation and Defibrillation

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Clinical Skills

Basic Life Support Part II: Airway/Breathing and Continued Cardiopulmonary Resuscitation
Julianna Jung 1
1The Johns Hopkins University School of Medicine

Basic Life Support Part II: Airway/Breathing and Continued Cardiopulmonary Resuscitation

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Clinical Skills

Intraosseous Needle Placement
Julianna Jung 1
1The Johns Hopkins University School of Medicine

Intraosseous Needle Placement

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Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)
Zach Hensel 1, Xiaona Fang 1,2,3, Jie Xiao 1
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , 3Department of Physics, Jilin University

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1.

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Biology

Super-resolution Imaging of the Bacterial Division Machinery
Jackson Buss 1, Carla Coltharp 1, Jie Xiao 1
1Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

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Medicine

A Preclinical Murine Model of Hepatic Metastases
Kevin C. Soares 1, Kelly Foley 2, Kelly Olino 1, Ashley Leubner 2, Skye C. Mayo 1, Ajay Jain 1, Elizabeth Jaffee 2, Richard D. Schulick 3, Kiyoshi Yoshimura 1,2, Barish Edil 1,2, Lei Zheng 1,2
1Department of Surgery, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 3Department of Surgery, University of Colorado Anschutz Medical Campus

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

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Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach
M. Natalia Vergara 1, Christian Gutierrez 1, M. Valeria Canto-Soler 1
1Wilmer Eye Institute, The Johns Hopkins University School of Medicine

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

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Neuroscience

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo
Mikko Juusola 1,2, An Dau 2, Lei Zheng 1, Diana Rien 1,2
1National Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, 2Department of Biomedical Science, The University of Sheffield

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

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Behavior

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice
Catherine M. Davis 1, Peter G. Roma 2, Robert D. Hienz 1
1Division of Behavioral Biology, Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 2Applied Behavior Biology Unit, Institutes for Behavior Resources

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

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Biology

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium
Peng Shang 1, Nadezda A. Stepicheva 1, Stacey Hose 1, J. Samuel Zigler, Jr. 2, Debasish Sinha 1,2
1Department of Ophthalmology, University of Pittsburgh School of Medicine, 2Wilmer Eye Institute, The Johns Hopkins University School of Medicine

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

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Chemistry

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules
Xin Guo 1, Xue-Mei Wang 1, Shou-Jun Xiao 1
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

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Cancer Research

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate
Shun Ishiyama 1,2,3,4, Casey Kissel 5, Xin Guo 1, Alexis Howard 6, Harumi Saeki 7, Tomoaki Ito 8, Polina Sysa-Shah 9, Hajime Orita 10, Kazuhiro Sakamoto 4, Kathleen Gabrielson 1,2
1Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 3Department of Surgery, Johns Hopkins University School of Medicine, 4Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 5Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, 6Tuskegee College of Veterinary Medicine, 7Department of Human Pathology, Juntendo University Faculty of Medicine, 8Department of Surgery, Juntendo University Shizuoka Hospital, Juntendo University School of Medicine, 9Department of Urology, The Johns Hopkins School of Medicine, 10Department of Gastroenterology and Minimally Invasive Surgery, Juntendo University Faculty of Medicine

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

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Biology

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes
Michelle Z. Chiu 1,2, Steven Lannon 1, Marisol Luchetti 1, Michael B. Wells 3, Deborah J. Andrew 1
1Department of Cell Biology, The Johns Hopkins University School of Medicine, 2Tufts School of Medicine, 3Department of Biomedical Sciences, Idaho College of Osteopathic Medicine

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

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Immunology and Infection

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells
Kevin C. Bermea 1, Sylvie T. Rousseau 1, Luigi Adamo 1
1Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

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Neuroscience

PyOKR: A Semi-Automated Method for Quantifying Optokinetic Reflex Tracking Ability
James K. Kiraly 1, Scott C. Harris 2, Timour Al-Khindi 1, Felice A. Dunn 2, Alex L. Kolodkin 1
1Solomon H. Snyder Department of Neuroscience, The Johns Hopkins Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, 2Department of Ophthalmology, University of California, San Franciso

We describe here PyOKR, a semi-automated quantitative analysis method that directly measures eye movements resulting from visual responses to two-dimensional image motion. A Python-based user interface and analysis algorithm allows for higher throughput and more accurate quantitative measurements of eye-tracking parameters than previous methods.

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