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17 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates
Mark A. Hughes 1, Paul M. Brennan 2, Andrew S. Bunting 3, Mike J. Shipston 1, Alan F. Murray 3
1Centre for Integrative Physiology, School of Biomedical Sciences, The University of Edinburgh, 2Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, 3School of Engineering, Institute for Integrated Micro and Nano Systems, The University of Edinburgh

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

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Neuroscience

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons
Sam A. Booker 1, Jie Song 1, Imre Vida 1
1Institute of Integrative Neuroanatomy, NeuroCure Cluster of Excellence, Charité Universitätmedizin

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

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Medicine

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration
Emily E. Hesketh 1, Alicja Czopek 1, Michael Clay 1, Gary Borthwick 1, David Ferenbach 1, David Kluth 1, Jeremy Hughes 1
1MRC Centre for Inflammation Research, University of Edinburgh

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

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Biology

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies
Samantha L. Eaton 1, Maica Llavero Hurtado 1, Karla J. Oldknow 2, Laura C. Graham 1, Thomas W. Marchant 1, Thomas H. Gillingwater 3,4, Colin Farquharson 2, Thomas M. Wishart 1,4
1Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 2Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, 3Centre for Integrative Physiology, University of Edinburgh, 4Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

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JoVE Journal

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin
Thomas Ramezani 1, Derek W. Laux 1, Isabel R. Bravo 1, Masazumi Tada 2, Yi Feng 1
1MRC Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, 2Department of Cell & Developmental Biology, University College London

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

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Medicine

Mouse Kidney Transplantation: Models of Allograft Rejection
George H. Tse *1, Emily E. Hesketh *1, Michael Clay 1, Gary Borthwick 1, Jeremy Hughes 1, Lorna P. Marson 1
1MRC Centre for Inflammation Research, The Queen's Medical Research Institute, The University of Edinburgh

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

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Medicine

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction
Emily E. Hesketh 1, Madeleine A. Vernon 1, Peng Ding 1, Spike Clay 1, Gary Borthwick 1, Bryan Conway 1, Jeremy Hughes 1
1MRC Centre for Inflammation Research, University of Edinburgh

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

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Developmental Biology

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification
Dean A. Houston *1, Katherine A. Staines *1, Vicky E. MacRae 1, Colin Farquharson 1
1Developmental Biology, The Roslin Institute and R(D)SVS, The University of Edinburgh

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

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Developmental Biology

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification
Cui Lin 1, Dongxing Zhu 2, Greg Markby 1, Brendan M. Corcoran 3, Colin Farquharson 1, Vicky E. Macrae 1
1Developmental Biology, The Roslin Institute and R(D)SVS, University of Edinburgh, 2Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, 3Clinical Sciences and R(D)SVS, University of Edinburgh

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

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Biology

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary
Wei Hu *1, Amin Tamadon *1, Aaron J.W. Hsueh 2, Yi Feng 1
1Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences; Institutes of Brain Science, Brain Science Collaborative Innovation Center, State Key Laboratory of Medical Neurobiology, Institute of Acupuncture and Moxibustion, Fudan Institutes of Integrative Medicine, Fudan University, 2Program of Reproductive and Stem Cell Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

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Neuroscience

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography
Fei Gao 1, Huibin Jia 2, Yi Feng 1
1Department of Pain Medicine, Peking University People's Hospital, 2Key Laboratory of Child Development and Learning Science of Ministry of Education, Research Center for Learning Science, School of Biological Sciences & Medical Engineering, Southeast University

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

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Neuroscience

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents
Sam A. Booker 1
1Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, The University of Edinburgh

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

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Genetics

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies
Natália Yumi Noronha *1, Guilherme da Silva Rodrigues *1, Marcela Augusta de Souza Pinhel 1, Jean-Baptiste Cazier 2,3, Lígia Moriguchi Watanabe 1, Albert Nobre Menezes 4, Carlos Roberto Bueno 5, Carolina Ferreira Nicoletti 1, Bruno Affonso Parenti de Oliveira 1, Isabelle Mello Schineider 6, Isabella Harumi Yonehara Noma 7, Igor Caetano Dias Alcarás 8, Fernando Barbosa 9, Carla Barbosa Nonino 6
1Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, 2Centre for Computational Biology, University of Birmingham, 3Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, 4Cancer Genetics and Evolution Laboratory, Cancer Research UK, Institute of Genetics & Molecular Medicine, The University of Edinburgh, 5Ribeirão Preto School of Physical Education and Sport, University of São Paulo, 6Health Sciences Department, Ribeirao Preto Medical School, University of Sao Paulo, 7Faculty of Pharmaceutical Sciences, University of São Paulo, 8Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, 9Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo

The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

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Cancer Research

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency
Amani Alshaikh 1,2, Dmytro Grygoryev 3, Dove Keith 4, Brett Sheppard 4,5, Rosalie C Sears 4,6, Jungsun Kim 3,6, Abdenour Soufi 1
1Centre for Regenerative Medicine, Institute of Regeneration and Repair, Institute of Stem Cell Research, The University of Edinburgh, 2King Abdulaziz City for Science and Technology Health Sector (KACST), 3Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, 4Brenden-Colson Canter for Pancreatic Care, Oregon Health & Science University, 5Department of Surgery, Oregon Health & Science University, 6Department of Molecular & Medical Genetics, Oregon Health & Science University

The present protocol describes the reprogramming of Pancreatic Ductal Adenocarcinoma (PDAC) and normal pancreatic ductal epithelial cells into induced pluripotent stem cells (iPSCs). We provide an optimized and detailed, step-by-step procedure, from preparing lentivirus to establishing stable iPSC lines.

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Immunology and Infection

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging
Sonia Elder 1, Justyna Cholewa-Waclaw 1, Elaine Emmerson 1
1The Centre for Regenerative Medicine, Institute for Regeneration and Repair, The University of Edinburgh

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

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Medicine

Regenerative Peripheral Nerve Interface: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain
Emily Pettersen 1,2,3,4, Paolo Sassu 5, Francesca Alice Pedrini 1,5, Hannes Granberg 1,2, Carina Reinholdt 2,6, Juan Manuel Breyer 7, Aidan Roche 8, Andrew Hart 9,10, Adil Ladak 11, Hollie A. Power 11, Michael Leung 12, Michael Lo 12, Ian Valerio 13, Kyle R. Eberlin 13, Jason Ko 14, Gregory A. Dumanian 14, Theodore A. Kung 15, Paul Cederna 15, Max Ortiz-Catalan 1,4,16,17
1Center for Bionics and Pain Research, 2Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, 3Department of Electrical Engineering, Chalmers University of Technology, 4Bionics Institute, 5IV Clinica Ortoplastica, IRCCS Istituto Ortopedico Rizzoli, 6Department of Hand Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, 7Department of Orthopedic Surgery, Hand Unit, Worker Hospital, 8College of Medicine and Veterinary Medicine, The Queen's Medical Research Institute, The University of Edinburgh, 9Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, 10College of Medicine, Veterinary & Life Sciences, The University of Glasgow, 11Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 12Plastics and Reconstructive Surgery, Dandenong Hospital, Monash Health, 13Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital & Harvard Medical School, 14Division of Plastic Surgery, Department of Surgery, Northwestern Feinberg School of Medicine, 15Section of Plastic Surgery, Department of Surgery, Michigan Medicine, 16Medical Bionics Department, University of Melbourne, 17Prometei Pain Rehabilitation Center

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular transposition.

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Medicine

Targeted Muscle Reinnervation: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain
Emily Pettersen 1,2,3,4, Paolo Sassu 5, Francesca Alice Pedrini 1,5, Hannes Granberg 1,2, Carina Reinholdt 2,6, Juan Manuel Breyer 7, Aidan Roche 8, Andrew Hart 9,10, Adil Ladak 11, Hollie A. Power 11, Michael Leung 12, Michael Lo 12, Ian Valerio 13, Kyle R. Eberlin 13, Theodore A. Kung 14, Paul Cederna 14, Jason M. Souza 15,16, Oskar Aszmann 17, Jason Ko 18, Gregory A. Dumanian 18, Max Ortiz-Catalan 1,4,19,20
1Center for Bionics and Pain Research, 2Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, 3Department of Electrical Engineering, Chalmers University of Technology, 4Bionics Institute, 5IV Clinica Ortoplastica, IRCCS Istituto Ortopedico Rizzoli, 6Department of Hand Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, 7Department of Orthopedic, Hand Unit, Worker Hospital, 8College of Medicine and Veterinary Medicine, The Queen's Medical Research Institute, The University of Edinburgh, 9Canniesburn Plastic Surgery Unit, Glasgow Royal Infirmary, 10College of Medicine, Veterinary & Life Sciences, The University of Glasgow, 11Division of Plastic Surgery, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 12Plastics and Reconstructive Surgery, Dandenong Hospital, Monash Health, 13Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital &, Harvard Medical School, 14Section of Plastic Surgery, Department of Surgery, Michigan Medicine, 15Department of Surgery, Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, 16Department of Plastic and Reconstructive Surgery, Ohio State University, 17Clinical Laboratory for Bionic Extremity Reconstruction, Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University Vienna, 18Division of Plastic Surgery, Department of Surgery, Northwestern Feinberg School of Medicine, 19Medical Bionics Department, University of Melbourne, 20Prometei Pain Rehabilitation Center

The protocol outlines the surgical procedure for the treatment of postamputation pain using Targeted Muscle Reinnervation (TMR). TMR will be compared with two other surgical techniques, specifically Regenerative Peripheral Nerve Interface (RPNI) and neuroma excision, followed by immediate burying within muscle under the context of an international, randomized controlled trial.

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