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61 ARTICLES PUBLISHED IN JoVE

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Biology

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model
Tobias Deuse 1, Fumiaki Ikeno 2, Robert C. Robbins 1, Sonja Schrepfer 1
1Department of Cardiothoracic Surgery, Stanford University School of Medicine, 2Stanford University School of Medicine

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

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Biology

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease
Xiaoqin Hua 1, Tobias Deuse 1,2, Karis R. Tang-Quan 1,2,3, Robert C. Robbins 3, Hermann Reichenspurner 1,2, Sonja Schrepfer 1,2,3
1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

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Biology

LAD-Ligation: A Murine Model of Myocardial Infarction
Mandy V.V. Kolk 1,2, Danja Meyberg 1,2, Tobias Deuse 1,2, Karis R. Tang-Quan 1,2,3, Robert C. Robbins 3, Hermann Reichenspurner 1,2, Sonja Schrepfer 1,2,3
1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

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Neuroscience

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow
Zaman Mirzadeh 1, Fiona Doetsch 2,3, Kazunobu Sawamoto 4, Hynek Wichterle 2,5, Arturo Alvarez-Buylla 1
1Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, 2Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, 3Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, 4Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, 5Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

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Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy
Mandy Stubbendorff 1, Tobias Deuse 1,2, Anna Hammel 1, Robert C. Robbins 2, Hermann Reichenspurner 1, Sonja Schrepfer 1,2
1University Heart Center Hamburg, Transplant and Stem Cell Immunobiology Lab (TSI), University Hospital Hamburg, 2Stanford University School of Medicine

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

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Biology

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation
Carissa Ritner 1, Harold S. Bernstein 1
1Cardiovascular Research Institute, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

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Biology

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development
Diane Hu 1, Ralph S. Marcucio 1
1Department of Orthopaedic Surgery, University of California San Francisco

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

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Medicine

Myo-mechanical Analysis of Isolated Skeletal Muscle
Peter E. Oishi 1,2, Sompob Cholsiripunlert 3, Wenhui Gong 2, Anthony J. Baker 4, Harold S. Bernstein 1,2,5
1Cardiovascular Research Institute, University of California San Francisco, 2Department of Pediatrics, University of California San Francisco, 3Department of Biology, San Francisco State University, 4Department of Medicine, University of California San Francisco , 5Eli and Edythe Broad Center of Regeneration Medicine & Stem Cell Research, University of California San Francisco

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

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Neuroscience

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain
Zhiqiang Dong 1, Mahendra Wagle 1, Su Guo 1
1Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences , University of California San Francisco

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

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Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)
Lenard Conradi 1,2, Christiane Pahrmann 1, Stephanie Schmidt 1, Tobias Deuse 1,3, Arne Hansen 2, Alexandra Eder 2, Hermann Reichenspurner 1, Robert C. Robbins 3, Thomas Eschenhagen 2, Sonja Schrepfer 1,3
1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

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Medicine

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects
Yan-yiu Yu 1, Chelsea Bahney 1, Diane Hu 1, Ralph S. Marcucio 1, Theodore Miclau, III 1
1Department of Orthopaedic Surgery, University of California, San Francisco

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

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Medicine

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis
Xiaoqin Hua 1,2, Tobias Deuse 1,2, Evangelos D. Michelakis 3, Alois Haromy 3, Phil S. Tsao 4, Lars Maegdefessel 4, Reinhold G. Erben 5, Claudia Bergow 5, Boris B. Behnisch 6, Hermann Reichenspurner 1,2, Robert C. Robbins 7, Sonja Schrepfer 1,2,7
1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine , 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

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Advanced Biology

Detection and Quantification of Nucleic Acids by Real Time PCR
Pablo Sanchez Bosch 2, Katja Brückner 1,2,3
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Department of Cell and Tissue Biology, University of California San Francisco, 3Cardiovascular Research Institute, University of California San Francisco

Detection and Quantification of Nucleic Acids by Real Time PCR

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Advanced Biology

Rapid Amplification of cDNA Ends
Pablo Sanchez Bosch 2, Sean Corcoran 2, Katja Brückner 1,2,3
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Department of Cell and Tissue Biology, University of California San Francisco, 3Cardiovascular Research Institute, University of California San Francisco

Rapid Amplification of cDNA Ends

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Biology

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection
Abrahim I. Orabi 1, Kamaldeen A. Muili 1, Dong Wang 1, Shunqian Jin 1, George Perides 2, Sohail Z. Husain 1
1Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, 2Department of Surgery, Tufts University Medical Center

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

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Biology

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus
Mark Slabodnick 1,2, Bram Prevo 1,3, Peter Gross 1,4, Janet Sheung 1,5, Wallace Marshall 1,2
1Physiology Course, Marine Biological Laboratory, 2Department of Biochemistry & Biophysics, University of California San Francisco, 3Department of Physics and Astronomy, Vrije Universiteit Amsterdam, 4Max Planck Institute of Molecular Cell Biology and Genetics, 5Department of Physics, University of Illinois Urbana-Champaign

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells. By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

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Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint
Andrew T. Jang 1, Jeremy D. Lin 1, Youngho Seo 2, Sergey Etchin 3, Arno Merkle 3, Kevin Fahey 3, Sunita P. Ho 1
1Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of California San Francisco, 2Department of Radiology and Biomedical Imaging, University of California San Francisco, 3Xradia Inc.

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e. geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

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Medicine

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models
Mandy Stubbendorff *1,2, Xiaoqin Hua *1,2, Tobias Deuse 1,2,3, Ziad Ali 4,5, Hermann Reichenspurner 2,3, Lars Maegdefessel 6, Robert C. Robbins 7, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, Cardiovascular Research Center, University Hospital Hamburg, 2Cardiovascular Research Center (CVRC) and DZHK University Hamburg, 3Department of Cardiovascular Surgery, University Heart Center Hamburg, 4Center for Interventional Vascular Therapy, Division of Cardiology, Columbia University, 5Cardiovascular Research Foundation, New York, 6Karolinska Institute, Stockholm, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine, Falk Cardiovascular Research Center

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

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Medicine

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model
Jing Feng 1, Yvonne Fitz 1, Yan Li 1, Melinda Fernandez 1, Irene Cortes Puch 1, Dong Wang 1, Stephanie Pazniokas 2, Brandon Bucher 3, Xizhong Cui 1, Steven B. Solomon 1
1Critical Care Medicine Department, Clinical Center, National Institutes of Health, 2Harvard Apparatus, 3ADInstruments

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

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Biology

Manipulating the Murine Lacrimal Gland
Jennifer K. Finley 1, D'Juan Farmer 1, Elaine Emmerson 1, Noel Cruz Pacheco 1, Sarah M. Knox 1
1Craniofacial and Mesenchymal Biology, Cell and Tissue Biology, University of California San Francisco

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

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Bioengineering

Production and Targeting of Monovalent Quantum Dots
Daeha Seo *1,2,3, Justin Farlow *4,5,6, Kade Southard 1,4,7, Young-wook Jun 1,7, Zev J. Gartner 4,5,6,7
1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

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Biology

Recovery of Adult Zebrafish Hearts for High-throughput Applications
Rima Arnaout 1, Sven Reischauer 2,3, Didier Y.R. Stainier 2,3
1Cardiovascular Research Institute and Division of Cardiology, University of California San Francisco, 2Department for Biochemistry and Biophysics, University of California San Francisco, 3Max-Planck Institute for Heart and Lung Research

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

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Developmental Biology

Assaying Blood Cell Populations of the Drosophila melanogaster Larva
Sophia Petraki 1, Brandy Alexander 1, Katja Brückner 1,2,3
1Department of Cell and Tissue Biology, University of California San Francisco, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 3Cardiovascular Research Institute, University of California San Francisco

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

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Developmental Biology

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies
Daniel Vogt 1, Pei-Rung Wu 1, Shawn F. Sorrells 2, Christine Arnold 2, Arturo Alvarez-Buylla 2, John L. R. Rubenstein 1
1Department of Psychiatry, University of California San Francisco, 2Department of Neurological Surgery, University of California San Francisco

GABAergic cortical interneuron progenitors disperse, develop and synaptically integrate into a host cortex after transplantation. These cells can be easily transduced before transplantation for in vivo studies of genetically modified GABAergic precursors. Here, we show viral labeling techniques to target specific interneuron subgroups using existing Cre lines and Cre-dependent reporters.

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Medicine

miRNA Expression Analyses in Prostate Cancer Clinical Tissues
Nathan Bucay 1, Varahram Shahryari 1, Shahana Majid 1, Soichiro Yamamura 1, Yozo Mitsui 1, Z. Laura Tabatabai 1, Kirsten Greene 1, Guoren Deng 1, Rajvir Dahiya 1, Yuichiro Tanaka 1, Sharanjot Saini 1
1Department of Urology, Veterans Affairs Medical Center, San Francisco, University of California San Francisco

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

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Medicine

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma
Aaron J. Clark 1, Shayan Fakurnejad 2, Quanhong Ma 2, Rintaro Hashizume 2,3
1Department of Neurological Surgery, University of California San Francisco, 2Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, 3Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

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Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device
Russell H. Cole 1, Tuan M. Tran 2, Adam R. Abate 3,4
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Joint UCSF/UCB Bioengineering Graduate Group, University of California, San Francisco, 3Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 4California Institute for Quantitative Biosciences, University of California, San Francisco

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

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Bioengineering

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers
Russell H. Cole 1, Zev J. Gartner 1, Adam R. Abate 2
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

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Medicine

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer
Varahram Shahryari 1, Hannah Nip 1, Sharanjot Saini 1, Altaf A. Dar 2, Soichiro Yamamura 1, Yozo Mitsui 1, Melissa Colden 1, Nathan Bucay 1, Laura Z. Tabatabai 1, Kirsten Greene 1, Guoren Deng 1, Yuichiro Tanaka 1, Rajvir Dahiya 1, Shahana Majid 1
1Department of Urology, VA Medical Center and UCSF, 2California Pacific Medical Center Research Institute

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

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Medicine

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency
Dong Wang 1,2,3,4, Grigol Tediashvili 1,2,3, Simon Pecha 4, Hermann Reichenspurner 4, Tobias Deuse 1,2,3,4, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, University Heart Center Hamburg, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research), partner site Hamburg/Kiel/Luebeck, 4Cardiovascular Surgery, University Heart Center Hamburg

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

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Developmental Biology

Live Imaging of Mouse Secondary Palate Fusion
Seungil Kim 1, Jan Prochazka 2, Jeffrey O. Bush 1
1Department of Cell and Tissue Biology, Program in Craniofacial Biology and Institute for Human Genetics, University of California San Francisco, 2Laboratory of Transgenic Models Diseases, Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

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Immunology and Infection

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4
Sharon A. Sagan *1,2, Andrés Cruz-Herranz *1, Collin M. Spencer 1,2, Peggy P. Ho 3, Lawrence Steinman 3, Ari J. Green 1, Raymond A. Sobel 4, Scott S. Zamvil 1,2
1Department of Neurology, University of California, 2Program in Immunology, University of California, 3Department of Neurology and Neurological Sciences, Stanford University, 4Department of Pathology, Stanford University

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

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Medicine

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta
Grigol Tediashvili 1,2,3, Dong Wang 1,2,3,4, Hermann Reichenspurner 4, Tobias Deuse 1,2,3,4, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Cardiovascular Surgery, University Heart Center

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

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Bioengineering

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies
Ana C. Silva 1,2,3,4, Maria J. Oliveira 1,2,5, Todd C McDevitt 4,6, Mário A. Barbosa 1,2,3, Diana S. Nascimento 1,2, Perpétua Pinto-do-Ó 1,2,3
1i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 3Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4Gladstone Institute of Cardiovascular Disease, 5Faculty of Medicine, University of Porto, 6University of California San Francisco

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

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Bioengineering

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
Benjamin Demaree 1,2, Daniel Weisgerber 1, Freeman Lan 1,2, Adam R. Abate 1,2,3
1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, 2UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, 3Chan Zuckerberg Biohub

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing >50,000 single-cell genomes from diverse cell populations.

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Developmental Biology

Methods for the Study of Regeneration in Stentor
Athena Lin 1, Tatyana Makushok 1, Ulises Diaz 1, Wallace F. Marshall 1
1Department of Biochemistry and Biophysics, University of California San Francisco

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

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Medicine

Cone Beam Intraoperative Computed Tomography-based Image Guidance for Minimally Invasive Transforaminal Interbody Fusion
Michael Safaee 1, Taemin Oh 1, Murat Pekmezci 2, Aaron J. Clark 1
1Department of Neurological Surgery, University of California, San Francisco, 2Department of Orthopedic Surgery, University of California, San Francisco

The purpose of this article is to provide image-guidance for minimally invasive transforaminal interbody fusion.

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Biochemistry

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
John S. Chorba 1, Adri M. Galvan 2, Kevan M. Shokat 2
1Division of Cardiology, Department of Medicine, Zuckerberg San Francisco General and University of California San Francisco, 2Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

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Medicine

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain
Nathaniel W. Jenkins 1, Claudia Iriondo 1,2, Vinil Shah 1, Emma Bahroos 1, Vahid Ravanfar 1,1, Melanie Regan 1, Youngho Seo 1, William P. Dillon 1, Sharmila Majumdar 1, Jason F. Talbott 1,3
1Radiology and Biomedical Imaging, University of California San Francisco, 2UCSF/UC Berkeley Graduate Program in Bioengineering, University of California San Francisco, 3Radiology and Biomedical Imaging, Zuckerberg San Francisco General Hospital

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

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Neuroscience

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats
Michael Dietrich 1, Christina Hecker 1, Alexander Hilla 2, Andrés Cruz-Herranz 3, Hans-Peter Hartung 1, Dietmar Fischer 2, Ari Green 3, Philipp Albrecht 1
1Department of Neurology, Heinrich-Heine-University Düsseldorf, 2Department of Cell Physiology, Faculty of Biology and Biotechnology, Ruhr-University Bochum, 3Division of Neuroinflammation and Glial Biology, Department of Neurology, University of California San Francisco

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

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Immunology and Infection

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Xiang Zhao 1, Maryam Hamidinia 1, Joanna Ai Ling Choo 1, Chien Tei Too 1,2, Zi Zong Ho 3, Ee Chee Ren 4, Antonio Bertoletti 3, Paul A. MacAry 1,2,5, Keith G. Gould 6, Joanna Brzostek 1, Nicholas R.J. Gascoigne 1,2,5
1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, 4Singapore Immunology Network, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 6Department of Immunology, Wright-Fleming Institute, Imperial College London

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

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Cancer Research

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Erin R. Bonner 1,2, Karim Saoud 1, Sulgi Lee 1,2, Eshini Panditharatna 3, Madhuri Kambhampati 1, Sabine Mueller 4,5, Javad Nazarian 1,2,5
1Center for Genetic Medicine, Children's National Health System, 2Institute for Biomedical Sciences, The George Washington University School of Medicine and Health Sciences, 3Dana-Farber Cancer Institute, 4Department of Neurology, University of California San Francisco, 5DIPG Centre of Expertise Zurich, Universitats-Kinderspital Zurich

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

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Neuroscience

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays
Jason E Chung *1,2, Hannah R Joo *1,2, Clay N Smyth 2, Jiang Lan Fan 3, Charlotte Geaghan-Breiner 2, Hexin Liang 2, Daniel Fan Liu 3, Demetris Roumis 2, Supin Chen 4,5, Kye Y Lee 4, Jeanine A Pebbles 4, Angela C Tooker 4, Vanessa M Tolosa 4,5, Loren M Frank 2,6
1Medical Scientist Training Program and Neuroscience Graduate Program, University of California San Francisco, 2Kavli Institute for Fundamental Neuroscience, Center for Integrative Neuroscience, and Department of Physiology, University of California San Francisco, 3Bioengineering Graduate Program, University of California San Francisco, 4Center for Micro- and Nanotechnology, Lawrence Livermore National Laboratory, 5Neuralink Corp., 6Howard Hughes Medical Institute

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

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Medicine

A Cryoinjury Model to Study Myocardial Infarction in the Mouse
Dong Wang *1,2, Grigol Tediashvili *1,2, Xiaomeng Hu 1,2, Alessia Gravina 2, Sivan G. Marcus 1,2, Hao Zhang 4, Jeffrey E Olgin 4, Tobias Deuse 1,2,5, Sonja Schrepfer 1,2,3,5
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco, 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Division of Cardiology, Cardiovascular Research Institute, University of California San Francisco, 5Cardiovascular Surgery, University Heart Center

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

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Neuroscience

Rapid Isolation of Dorsal Root Ganglion Macrophages
Xiaobing Yu 1, Jacqueline Leff 1, Zhonghui Guan 1
1Department of Anesthesia and Perioperative Care, University of California San Francisco

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

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Bioengineering

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics
Jonathon M. Muncie 1,2, Roberto Falcón-Banchs 1, Johnathon N. Lakins 2, Lydia L. Sohn 1,3, Valerie M. Weaver 2,4,5,6
1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California San Francisco, 3Department of Mechanical Engineering, University of California Berkeley, 4Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 5UCSF Comprehensive Cancer Center, Helen Diller Family Cancer Research Center, University of California San Francisco, 6Department of Anatomy, Department of Bioengineering and Therapeutic Sciences, and Department of Radiation Oncology, University of California San Francisco

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

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Cancer Research

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients
Divya Bhagirath 1, Rajvir Dahiya 2, Shahana Majid 2, Z. Laura Tabatabai 3, Sharanjot Saini 1
1Department of Biochemistry and Molecular Biology, Augusta University, 2Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, 3Department of Pathology, Veterans Affairs Medical Center and University of California, San Francisco

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

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Medicine

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples
Andrew Reckers 1, Anita Wen 1, David Aguilar 1, Peter Bacchetti 2, Monica Gandhi 3, John Metcalfe 4, Roy Gerona 1
1Department of Obstetrics, Gynecology and Reproductive Sciences, TB Hair Analysis Laboratory, University of California San Francisco, 2Department of Epidemiology and Biostatistics, University of California San Francisco, 3Department of Medicine, Division of HIV, Infectious Diseases, and Global Medicine, University of California San Francisco, 4Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California San Francisco

Current methods of analyzing patients’ adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

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Bioengineering

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing
Jesse Q. Zhang 1,2, Kai-Chun Chang 1, Leqian Liu 1, Zev J. Gartner 3,5, Adam R. Abate 1,4,5
1Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 2University of California Berkeley-UCSF Graduate Program in Bioengineering, University of California San Francisco, 3Department of Pharmaceutical Chemistry, University of California San Francisco, 4California Institute for Quantitative Biosciences, University of California San Francisco, 5Chan Zuckerberg Biohub

A bottleneck in the ‘design-build-test’ cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

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Developmental Biology

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer
Ulises Diaz 1,2, Wallace Marshall 2, Blake Riggs 1
1Department of Biology, San Francisco State University, 2Department of Biochemistry & Biophysics, UCSF Mission Bay

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

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Bioengineering

Simple, Affordable, and Modular Patterning of Cells using DNA
Katelyn A. Cabral 1, David M. Patterson 2, Olivia J. Scheideler 1, Russell Cole 3, Adam R. Abate 4,5,6, David V. Schaffer 7,8, Lydia L. Sohn 9, Zev J. Gartner 2,6,10
1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Department of Pharmaceutical Chemistry, University of California San Francisco, 3Scribe Biosciences, 4Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 5California Institute for Quantitative Biosciences, University of California San Francisco, 6Chan Zuckerberg Biohub, University of California San Francisco, 7Department of Chemical & Biomolecular Engineering, University of California Berkeley, 8Helen Wills Neuroscience Institute, University of California Berkeley, 9Department of Mechanical Engineering, University of California Berkeley, 10Center for Cellular Construction, University of California San Francisco

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

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Biology

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays
Daniel W. Weisgerber 1, Makiko N. Hatori 1, Adam R. Abate 1,2
1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, 2Chan Zuckerberg Biohub

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

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Biology

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking
Janet Y. Sheung 1, Megan Otsuka 2, Gabriella Seifert 3, Athena Lin 4, Wallace F. Marshall 4
1W. M. Keck Science Department, Scripps, Pitzer, and Claremont McKenna of The Claremont Colleges, 2W. M. Keck Science Department, Pitzer College, 3W. M. Keck Science Department, Scripps College, 4Department of Biochemistry and Biophysics, School of Medicine, University of California at San Francisco

We present a protocol for the characterization of motility and behavior of a population of hundred micron- to millimeter-sized cells using brightfield microscopy and cell tracking. This assay reveals that Stentor coeruleus transitions through four behaviorally distinct phases when regenerating a lost oral apparatus.

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Neuroscience

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice
Liangliang He *1,2, Wenxing Zhao *1, Lingyi Zhang 2, Maalveka Ilango 2,3, Na Zhao 2, Liqiang Yang 1, Zhonghui Guan 2
1Department of Pain Management, Xuanwu Hospital, Capital Medical University, 2Department of Anesthesia and Perioperative Care, University of California San Francisco, 3University of Washington

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

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Medicine

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China
Cheng Luo *1, Chengming Fan *1, Hao Zhang 1, Long Song 1, Yuhong Liu 1, Liming Liu 1
1Department of Cardiovascular Surgery, the Second Xiangya Hospital, Central South University

We summarize the Cox-Maze IV procedure concomitant with valvular surgery performed in patients with situs inversus dextrocardia at this institution.

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Cancer Research

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer
Irene Chicote 1, Jordi Martínez-Quintanilla 1, Juan Antonio Cámara 2, Héctor G. Palmer 1,3
1Stem Cells and Cancer Laboratory, Vall d’Hebron Institute of Oncology (VHIO), 2Preclinical Therapeutics Core, University of California San Francisco, 3CIBERONC

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

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Biology

Studying Habituation in Stentor coeruleus
Deepa Rajan 1, Peter Chudinov 1, Wallace Marshall 1
1Department of Biochemistry and Biophysics, University of California San Francisco

We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations.

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Cancer Research

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids
Olivia Graham *1, Jeimmy Rodriguez *1, Lillian van Biljon 1, Bisiayo Fashemi 1, Emily Graham 1, Katherine Fuh 1,2, Dineo Khabele 1, Mary Mullen 1
1Washington University in St. Louis, 2University of California San Francisco

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

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Cancer Research

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays
Lillian van Biljon 1, Bisiayo Fashemi 1, Jeimmy Rodriguez 1, Olivia Graham 1, Amanda Compadre 1, Katherine Fuh 1,2, Dineo Khabele 1, Mary Mullen 1
1Washington University in St. Louis, 2University of California San Francisco

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

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Developmental Biology

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors
Xiang Zhao 1,2, Su Guo 2,3
1Chan Zuckerberg Biohub, 2Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 3Programs in Human Genetics and Biological Sciences, University of California San Francisco

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

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Neuroscience

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning
Liangliang He *1,2, Wenxing Zhao *1, Lingyi Zhang 2,3, Maalveka Ilango 2,4, Na Zhao 2, Liqiang Yang 1, Zhonghui Guan 2
1Department of Pain Management, Xuanwu Hospital, Capital Medical University, 2Department of Anesthesia and Perioperative Care, University of California San Francisco, 3Department of Anesthesiology, Sun Yat-Sen University, 4University of Washington

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

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