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22 ARTICLES PUBLISHED IN JoVE

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Biology

Drosophila Pupal Abdomen Immunohistochemistry
Wei Wang 1, John H. Yoder 1
1Department of Biological Sciences, University of Alabama

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

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Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window
Lifeng Zhang *1, Andree Lapierre *1, Brittany Roy 1, Maili Lim 1, Jennifer Zhu 1, Wei Wang 1, Stephen B. Sampson 1, Kyuson Yun 1, Bonnie Lyons 1, Yun Li 1, Da-Ting Lin 1
1The Jackson Laboratory

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

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Neuroscience

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
Yun Li 1, Brittany D. Roy 1, Wei Wang 1, Lifeng Zhang 1, Stephen B. Sampson 1, Da-Ting Lin 1
1The Jackson Laboratory

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

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Chemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization
Wei Wang 1, John M. Lester 1, Anthony E. Amorosa 2, Deborah L. Chance 3, Valeri V. Mossine 1, Thomas P. Mawhinney 1,4
1Department of Biochemistry, University of Missouri, 2Department of Biological Sciences, University of Missouri, 3Department of Molecular Microbiology & Immunology, University of Missouri, 4Department of Child Health, University of Missouri

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

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Neuroscience

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice
Xiaojing Lin 1,2, Tingbao Zhao 3, Wenhui Xiong 1, Shaonan Wen 4, Xiaoming Jin 1, Xiao-Ming Xu 1
1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Department of Anatomy and Cell Biology, Indiana University School of Medicine, 2Department of Spinal Cord Injury and Repair, Trauma and Orthopedics Institute of Chinese PLA, General Hospital of Jinan Military Region, 3Department of Orthopedics, Shandong Cancer Hospital, Shandong University, 4Department of Neurobiology, Institute of Basic Medical Sciences, Academy Military Medical Sciences

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

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Medicine

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats
Xiao-Jing Lin 1,2,3, Shaonan Wen 2,6, Ling-Xiao Deng 3, Heqiao Dai 3, Xiaolong Du 3, Chen Chen 3,4, Melissa J. Walker 3,4, Ting-Bao Zhao 1,5, Xiao-Ming Xu 3
1Department of Spinal Cord Injury and Repair, Trauma and Orthopedics Institute of Chinese PLA, 960th Hospital, Joint Logistics Support Force of PLA, 2Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Academy of Military Sciences, 3Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman Campbell Brain and Spine, Indiana University School of Medicine, 4Program in Medical Neuroscience, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, 5Department of Orthopedics, the Third Affiliated Hospital, Shandong University, 6Beijing Computing Center, Beijing Academy of Science and Technology

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

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Chemistry

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering
Jinpeng Wu 1,2, Shawn Sallis 2,3, Ruimin Qiao 2, Qinghao Li 2,4, Zengqing Zhuo 2,5, Kehua Dai 2,6, Zixuan Guo 2,7, Wanli Yang 2
1Geballe Laboratory for Advanced Materials, Stanford University, 2Advanced Light Source, Lawrence Berkeley National Laboratory, 3Department of Materials Science and Engineering, Binghamton University, 4School of Physics, National Key Laboratory of Crystal Materials, Shandong University, 5School of Advanced Materials, Peking University Shenzhen Graduate School, 6School of Metallurgy, Northeastern University, 7Department of Chemical Engineering, University of California-Santa Barbara

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

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Developmental Biology

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue
Zhenan Liu 1, Jie Wen 1,2, Xue Leng 1, Qian Zhou 1, Changkuo Zhou 3, Huaqiang Zhao 1, Xunwei Wu 1,2
1Shandong Provincial Key Laboratory of Oral Tissue Regeneration and Laboratory for Tissue Engineering and Regeneration, School of Stomatology, Shandong University, 2Suzhou Institute of Shandong University, 3Department of Urology, Qilu Hospital of Shandong University

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

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Medicine

A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts
John Harry Caufield 1,2, David A. Liem 1,2,3, Anders O. Garlid 1,2, Yijiang Zhou 4, Karol Watson 1,3, Alex A. T. Bui 1,5,6,7, Wei Wang 1,7,8,9, Peipei Ping 1,2,3,7,8
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Medicine/Cardiology, University of California, Los Angeles, 4Department of Cardiology, First Affiliated Hospital, Zhejiang University School of Medicine, 5Department of Radiological Sciences, University of California, Los Angeles, 6Department of Bioengineering, University of California, Los Angeles, 7Scalable Analytics Institute (ScAi), University of California, Los Angeles, 8Department of Bioinformatics, University of California, Los Angeles, 9Department of Computer Science, University of California, Los Angeles

We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

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Medicine

Cloud-Based Phrase Mining and Analysis of User-Defined Phrase-Category Association in Biomedical Publications
Dibakar Sigdel *1,2, Vincent Kyi *1,2, Aiden Zhang *1, Shaun P. Setty 3, David A. Liem 1,2,4, Yu Shi 5, Xuan Wang 5, Jiaming Shen 5, Wei Wang 1,6,7, JiaWei Han 5, Peipei Ping 1,2,4,6
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Pediatric and Adult Congenital Heart Surgery, Miller Children's and Women's Hospital and Long Beach Memorial Hospital, 4Department of Medicine/Cardiology, University of California, Los Angeles, 5NIH BD2K Program Centers of Excellence for Big Data Computing -- KnowEng Center, Department of Computer Science, University of Illinois at Urbana-Champaign (UIUC), 6Scalable Analytics Institute (ScAi), University of California, Los Angeles, 7Department of Computer Science, University of California, Los Angeles

We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain.

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Medicine

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach
Ali R. Ahmadi 1, Le Qi 1, Kenichi Iwasaki 1, Wei Wang 1, Russell N. Wesson 1, Andrew M. Cameron 1, Zhaoli Sun 1
1Department of Surgery, Johns Hopkins University School of Medicine

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

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Environment

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System
Weitao Hou 1, Hanpeng Wang 1, Wei Wang 1, Zhongzhong Liu 1, Qingchuan Li 1
1Research Center of Geotechnical and Structural Engineering, Shandong University

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal’s physical and mechanical properties induced by CO2 adsorption.

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Medicine

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Di Liu *1, Xizhu Xu *2, Yuejin Li 2, Jie Zhang 1, Xiaoyu Zhang 1, Qihuan Li 1, Haifeng Hou 2, Dong Li 2, Wei Wang 1,2,3, Youxin Wang 1
1Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, 2School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, 3School of Medical and Health Sciences, Edith Cowan University

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

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Immunology and Infection

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction
Hongzheng Bi 1,2, Samiksha Wasnik 1, David J. Baylink 1, Chenfan Liu 1,3,4, Xiaolei Tang 1,3
1Division of Regenerative Medicine, Department of Medicine, Loma Linda University, 2Zhengzhou University, 3Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, Long Island University, 4Jinan Infectious Disease Hospital, Shandong University

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

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Biology

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues
Chang Liu 1, Shuangshuang Wang 1, Man Liu 2, Fuxiang Bai 1, Zhihong Chen 3, Ping Wang 4, Jun Mi 1,5, Xunwei Wu 1
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Shijiazhuang Shimen Experimental School, 3Qilu Children's Hospital of Shandong University, 4Qilu Hospital of Shandong University, 5Shenzhen Research Institute of Shandong University

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

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Behavior

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice
Yi Xie 1, Sai-Yue Deng 1, Si-Miao Chen 1, Xue-Jiao Chen 1, Wen-Wen Lai 1, Li-Fang Huang 1, Li Ba 1, Wei Wang 1,2, Feng-Fei Ding 1,3
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China, 2Key Laboratory of Neurological Diseases of Chinese Ministry of Education, The School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000,China, 3Department of Pharmacology, School of Basic Medical Science; State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

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Neuroscience

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method
Weiyan Jiang *1, Congcong Jin *2, Weiming Xu *3, Yingxian Li 4, Yinghong Lin 5, Shengxiang Liang 6,7, Wei Wang 8
1Department of Clinical Laboratory, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 2Rehabilitation Department, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, 3Department of Neurosurgery, the Hospital of Changle, 4Pediatrics Ward 2, Shouguang Hospital of Traditional Chinese Medicine, 5Fujian University of Traditional Chinese Medicine, 6National-Local Joint Engineering Research Center of Rehabilitation Medicine Technology, Fujian University of Traditional Chinese Medicine, 7Rehabilitation Industry Institute, Fujian University of Traditional Chinese Medicine, 8Department of Neurosurgery, the First Affiliated Hospital of Wenzhou Medical University

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

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Biology

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids
Jiabei Lian *1,2, Xinyuan Meng *1,3, Xiaohui Zhang 1,2, Huili Hu 1,2
1Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, School of Basic Medical Sciences, Stem Cell and Regenerative Medicine Research Center, Cheeloo College of Medicine, Shandong University, 2The Research Center of Stem Cell and Regenerative Medicine, Shandong University Cheeloo Medical College, School of Basic Medical Sciences, Shandong University

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

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Bioengineering

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632
Zhiwei Xie 1,2,3, Jizhou Shi 4, Min Zong 3, Qiuping Xu 1, Chang Liu 1, Jie Wen 1, Qun Zhang 1, Panpan Liu 1, Guanyi Liu 1, Jing Guo 2,5, Xunwei Wu 1
1Department of Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 2Department of Orthodontics, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, 3Department of Stomatology, Shengli Oilfield Central Hospital, 4Department of Pediatric Surgery, Shengli Oilfield Central Hospital, 5Ningbo Stomatology Hospital of Savaid Stomatology School, Hangzhou Medical College

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

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Neuroscience

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives
Kristina Xiao Liang *1,2, Anbin Chen *1,2,3,4, Cecilie Katrin Kristiansen 1,2, Laurence A. Bindoff 1,2
1Department of Clinical Medicine (K1), University of Bergen, 2Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Haukeland University Hospital, 3Department of Neurosurgery, Qilu Hospital and Institute of Brain and Brain-Inspired Science, Cheeloo College of Medicine, Shandong University, 4Shandong Key Laboratory of Brain Function Remodeling

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

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Medicine

Extraperitoneal Laparoscopic Modified Y-V Plasty for the Treatment of Refractory Bladder Neck Contracture
Zhenjie Zang 1, Dingchang Shao 1, Hui Zhang 2, Dingqi Sun 2, Tongxiang Diao 2, Keqin Zhang 2, Qiang Fu 1,2
1Department of Urology, Shandong Provincial Hospital, Shandong University, 2Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University

Here, we describe a modified surgical procedure for refractory bladder neck contracture that is associated with easy manipulation, optimum intraoperative exposure, and minimal invasiveness.

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Biology

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model
Yanjing Guo 1,2,3, Wei Li 2,3, Yinghan Wang 2
1The Affiliated Hospital of Qingdao University, 2Department of Anesthesiology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 3Department of Anesthesiology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

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