JoVE General
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.
JoVE General
Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution
1Department of Cellular and Molecular Medicine, University of California, San Diego, 2Biosecurity and Public Health, Los Alamos National Laboratory
Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.
JoVE General
Methods for the Study of the Zebrafish Maxillary Barbel
1Department of Biological Sciences, DePaul University, 2Children’s Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine
The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.
JoVE Immunology and Infection
Granulocyte-dependent Autoantibody-induced Skin Blistering
1Department of Dermatology, University of Freiburg, 2Kepler High School Freiburg, 3Centre for Biological Signalling Studies (BIOSS), University of Freiburg
In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.
JoVE Clinical and Translational Medicine
Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens
1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center
Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed “tumor explant method”. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.
JoVE Immunology and Infection
Induction of Experimental Autoimmune Hypophysitis in SJL Mice
Department of Pathology, The Johns Hopkins University
This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.
JoVE Bioengineering
In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage
1Neurosurgery, Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center, 3Molecular & Cellular Biology, Baylor College of Medicine
We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.
JoVE Immunology and Infection
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University
This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).
JoVE Clinical and Translational Medicine
Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
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.
JoVE General
Laser Capture Microdissection of Mammalian Tissue
Department of Pathology, University of California, Irvine (UCI)
JoVE General
Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles
Gene Expression and Signaling Research Group, Max Planck Institute for Biophysical Chemistry
Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.
JoVE Immunology and Infection
Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model
Chagas Disease Multidisciplinary Research Laboratory, University of Brasilia
The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.
JoVE General
Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells
Department of Pathology, University of Wisconsin - Milwaukee
Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.
JoVE Clinical and Translational Medicine
Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy
1Raymond and Beverly Sackler Foundation, 2The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 3School of Natural Sciences, Institute for Advanced Study, Princeton, New Jersey
We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.
JoVE Immunology and Infection
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
JoVE General
RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs
Department of Biological Sciences, Union College
By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.
JoVE Immunology and Infection
Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches
Division of Infectious Diseases, New York State Department of Health
There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.
JoVE Neuroscience
Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex
1Department of Molecular, Cellular Biology and Biochemistry, Brown University, 2Institute for Brain Science, Brown University, 3Department of Psychiatry and Human Behavior, Warren Alpert School of Medicine, Brown University
To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.
JoVE Clinical and Translational Medicine
The Polyvinyl Alcohol Sponge Model Implantation
1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 2The Department of Veterans Affairs Medical Center, 3Internal Medicine, Vanderbilt University School of Medicine
A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.
JoVE General
Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University
The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.
JoVE General
A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt
1Department of Biology, University of Dayton, 2Center for Tissue Regeneration and Engineering, University of Dayton
In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.
JoVE General
Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs
Department of Physiology and Biophysics, University of California, Irvine (UCI)
JoVE General
Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation
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.
JoVE Clinical and Translational Medicine
Mouse Eye Enucleation for Remote High-throughput Phenotyping
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University
The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.
JoVE Clinical and Translational Medicine
Ex Vivo Infection of Live Tissue with Oncolytic Viruses
Center for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI)
Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.
JoVE General
Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University
Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.
JoVE Bioengineering
The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression
Molecular and Cellular Oncogenesis Program, The Wistar Institute
In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.
JoVE Immunology and Infection
Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)
Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
JoVE Clinical and Translational Medicine
Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)
Division of Gastroenterology, Washington University School of Medicine
We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.
JoVE General
Plastic Embedding and Sectioning of Xenopus laevis Embryos
1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2University of California, Irvine (UCI)
Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.
JoVE Clinical and Translational Medicine
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
1Department of Preventive Medicine, University of Southern California, 2Institute for Women's Health, University College London
We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.
JoVE General
In Situ Hybridization for the Precise Localization of Transcripts in Plants
The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.
JoVE Immunology and Infection
Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis
1Division of Microbiology, Tulane National Primate Research Center, 2Division of Comparative Pathology, Tulane National Primate Research Center, 3Division of Veterinary Medicine, Tulane National Primate Research Center
Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.
JoVE Clinical and Translational Medicine
Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice
1European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, 2British Heart Foundation Cardiovascular Sciences Unit, Imperial College London, 3Department of Bioengineering, Imperial College London, 4Biomedical Engineering, Eindhoven University of Technology
The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.
JoVE General
LAD-Ligation: A Murine Model of Myocardial Infarction
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.
JoVE Bioengineering
Skin Tattooing As A Novel Approach For DNA Vaccine Delivery
1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 2Department of Pathology, New York University School of Medicine, 3Healthcare System, Veterans Affairs New York Harbor
Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.
JoVE General
Isolation and Analysis of Hematopoietic Stem Cells from the Placenta
Jonsson Comprehensive Cancer Center, University of California, Los Angeles
We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.
JoVE General
Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization
1Department of Cancer Genetics, BC Cancer Research Centre, 2Deeley Research Centre, BC Cancer Agency, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency
This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.
JoVE Clinical and Translational Medicine
Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence
1Division of Pathology, University of Edinburgh, 2HistoRx Inc.
Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.
JoVE Clinical and Translational Medicine
Performing and Processing FNA of Anterior Fat Pad for Amyloid
1Department of Pathology, Medical College of Wisconsin, 2Current Address: Department of Pathology, Wayne State University School of Medicine Detroit Medical Center, 3Department of Neurology, Medical College of Wisconsin, 4Department of Medicine, Medical College of Wisconsin, 5Division of Neoplastic Diseases and Related Disorders, Medical College of Wisconsin
Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.
JoVE General
Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos
Department of Genetics, University of Georgia
This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.
JoVE General
Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)
1Department of Cancer Biology, Vanderbilt University, 2Departments of Medicine and Cancer Biology, Vanderbilt University
Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.
JoVE Neuroscience
Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis
1Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Department of Neurological Surgery, University of California, San Francisco, 3Department of Neurology, University of California, San Francisco
In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.
JoVE Immunology and Infection
Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut
1The Biomedical Research Centre, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia
Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.
JoVE General
Evaluation of Mammary Gland Development and Function in Mouse Models
Department of Anatomy and Cell Biology, University of Western Ontario
This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.
JoVE Clinical and Translational Medicine
Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury
1Department of Anesthesiology and Perioperative Medicine, Oregon Health & Sciences University, 2Department of Pharmacology, University of Colorado Denver
A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.
JoVE Immunology and Infection
Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
1Institute of Immunology, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Pathobiology, School of Veterinary Medicine, University of Pennsylvania
Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.
JoVE General
Bioengineering Human Microvascular Networks in Immunodeficient Mice
Department of Cardiac Surgery, Children's Hospital Boston, Harvard Medical School
Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.
JoVE General
Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia
A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.
JoVE Immunology and Infection
Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis
Louisiana State University Health Sciences Center
Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.
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