JoVE Application Notes
1Cell Signaling Technology
Immunohistochemistry (IHC) is a technique used to analyze protein expression in the context of tissue morphology. This article describes an IHC protocol optimized by scientists at Cell Signaling Technology, for use with our antibodies, that you can replicate to obtain the best results in your experiments.
Published April 10, 2014. Keywords: Immunohistochemistry, IHC, paraffin, SignalStain, antibody, DAB, unmasking, antigen retrieval, Cell Signaling Technology, CST.
1Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry
Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.
Published September 2, 2013. Keywords: Chemistry, Clinical Chemistry Tests, Proteomics, Proteomics, Proteomics, analytical chemistry, Formalin fixed and paraffin embedded (FFPE), sample preparation, proteomics, filter aided sample preparation (FASP), clinical proteomics; microdissection, SAX-fractionation
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.
Published September 21, 2012. Keywords: Medicine, Cellular Biology, Molecular Biology, Immunology, Biomedical Engineering, mucus, lectins, OCT, imaging, sialic acids, glycosylation
1Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 2Department of Pathology and Laboratory Medicine, Indiana University Health
This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.
Published July 9, 2014. Keywords: Genetics, qPCR, cytomegalovirus, CMV, biopsy, real-time PCR, gastrointestinal, formalin-fixed, paraffin-embedded tissue
JoVE Immunology and Infection
1Department of Oral Microbiology and Immunology, School of Dentistry and Dental Research Institute, Seoul National University
Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.
Published May 21, 2015. Keywords: Immunology, periodontology, oral microbiology, in situ hybridization, 16S rRNA, bacteria, paraffin-embedded tissue, species-specific, universal
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.
Published March 26, 2011. Keywords: Genetics, DNA extraction, paraffin embedded tissue, phenol:chloroform extraction, genetic analysis, epigenetic analysis
1Institute of Pathology, University Medical Center, Göttingen, 2Department of Hematology/Oncology, Goethe University of Frankfurt, 3Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 4Bioanalytics, Institute of Clinical Chemistry, University Medical Center, Göttingen, 5German Cancer Consortium, 6German Cancer Research Center
In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.
Published February 27, 2015. Keywords: Medicine, Proteomics, solid tumors, leukemia, formalin-fixed and paraffin-embedded tissue (FFPE), laser-capture microdissection, spike-in SILAC, quantitative mass spectrometry
1Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole polytechnique fédérale de Lausanne
We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.
Published January 8, 2015. Keywords: Medicine, Hormone signaling, breast cancer, reduction mammoplasty, breast tissue microstructures, ex vivo model, estrogen, progesterone, mammary epithelial cells, tissue digestion, paracrine signaling, microenvironment, tissue architecture
1Pediatric Diabetes Research Center, Department of Pediatrics, University of California, San Diego
A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described. The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.
Published May 18, 2014. Keywords: Medicine, human fetal pancreas, islet cell cluster (ICC), transplantation, immunofluorescence, endocrine cell proliferation, differentiation, C-peptide
1Department of Leukemia, MD Anderson Cancer Center, 2Wake Forest Baptist Medical Center, Institute for Regenerative Medicine
Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.
Published September 22, 2013. Keywords: Medicine, Immunology, Medicine, Cellular Biology, Molecular Biology, Genetics, Anatomy, Physiology, Biomedical Engineering, Immunohistochemistry (IHC), Microscopy, Fluorescence, Regeneration, Cellular Microenvironment, Tumor Microenvironment, Cell Biology, Investigative Techniques, Biological Phenomena, Mesenchymal stem cells (MSC), Tumor/Cancer associated fibroblasts (TAF/CAF), transgenic mouse model, regenerative medicine, wound healing, cancer
JoVE Immunology and Infection
1Bacteriology and Parasitology, Tulane National Primate Research Center, 2Microbiology and Immunology, Tulane National Primate Research Center
Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.
Published June 5, 2014. Keywords: Immunology, Microdissection, mesodissection, formalin fixed paraffin embedded, Mtb, LCM, TB, Mycobacterium tuberculosis
1Department of Child Health, University of Arizona College of Medicine - Phoenix, 2BARROW Neurological Institute, Phoenix Children's Hospital, 3Department of Biology and Biochemistry, University of Bath, 4Neuroscience Program, Arizona State University, 5Phoenix VA Healthcare System
This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.
Published May 12, 2015. Keywords: Neuroscience, Immunohistochemistry, Iba1, Pan-Neuronal, Fluorescence, Double-Labeling, Microglia, Neuron, Rat, Antibody
1Advanced Cell Diagnostics, Inc.
The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.
Published March 11, 2014. Keywords: Medicine, RNAscope, Head and Neck Squamous Cell Carcinoma (HNSCC), Oropharyngeal Squamous Cell Carcinoma (OPSCC), Human Papillomavirus (HPV), E6/ E7 mRNA, in situ hybridization, tumor
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.
Published July 29, 2011. Keywords: Medicine, Glioblastoma multiforme, glioma, temozolomide, therapeutics, drug design
1Department of Bioengineering, University of Illinois at Chicago, 2Department of Pathology, University of Illinois at Chicago, 3Department of Biological Sciences, University of Illinois at Chicago, 4Department of Chemistry, University of Illinois at Chicago, 5Department of Nephrology, University of Illinois at Chicago
Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.
Published January 21, 2015. Keywords: Medicine, Spectroscopy, Imaging, Fourier Transform, Pathology, Cancer, Liver, Kidney, Hyperspectral, Biopsy, Infrared, Optics, Tissue
1Cancer Sciences Unit, University of Southampton School of Medicine, 2University Surgical Unit, University of Southampton School of Medicine, 3Bioinformatics Unit, London Research Institute, Cancer Research UK
Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells.
Published April 29, 2014. Keywords: Medicine, Colorectal cancer, Cancer metastasis, organotypic culture, laser microdissection, molecular profiling, invasion, tumor microenvironment, stromal tissue, epithelium, fibroblasts
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.
Published October 11, 2014. Keywords: Medicine, transplantation, mouse model, surgery, kidney, immunology, rejection
1Department of Neurology, Vanderbilt University Medical Center, 2Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, 3Neurology Service, Veteran Affairs TVHS
Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.
Published January 14, 2014. Keywords: Medicine, Glioma, Malignant glioma, primary orthotopic xenograft, isocitrate dehydrogenase
JoVE Immunology and Infection
1Chagas 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.
Published July 29, 2012. Keywords: Immunology, Infection, Genetics, Parasitology, Trypanosoma cruzi, Gallus gallus, transfer of mitochondrial kDNA minicircle, targeted-prime TAIL-PCR, genotype modifications, Chagas disease
1Department of Cancer Biology, UT MD Anderson Cancer Center, 2Novartis Institutes for Biomedical Research, 3Sanofi US, 4Institute of Applied Cancer Science, UT MD Anderson Cancer Center
Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.
Published April 13, 2015. Keywords: Medicine, Telomerase, Telomere, mTERT-ER, LSL-mTERT, Ageing, Cancer, Neural Stem Cells
1Baylor College of Medicine, 2Michael E. DeBakey Veterans Affairs Medical Center
We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.
Published May 27, 2014. Keywords: Medicine, Brain Ischemia, Molecular Probe Techniques, Investigative Techniques, experimental stroke, focal brain ischemia, 5'OH DNA breaks, phagocytic clearance, in situ detection, phagocytosis labeling, DNA damage
1Department of Orthopaedic Surgery and Traumatology, Ghent University Hospital, 2Department of Radiation Oncology and Experimental Cancer Research, Ghent University, 3Department of Virology, Parasitology, and Immunology, Ghent University, 4Pathlicon
The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.
Published July 17, 2013. Keywords: Cancer Biology, Medicine, Cellular Biology, Molecular Biology, Biomedical Engineering, Bioengineering, Developmental Biology, Anatomy, Physiology, Oncology, Surgery, Adipose Tissue, Connective Tissue, Neoplasm, Muscle Tissue, Sarcoma, Animal Experimentation, Cell Culture Techniques, Neoplasms, Experimental, Neoplasm Transplantation, Biological Assay, Sarcomas, CAM-assay, CAM, assay, xenograft, proliferation, invasion, cancer, tumor, in ovo, animal model
JoVE Immunology and Infection
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.
Published October 12, 2012. Keywords: Immunology, Medicine, Physiology, Anatomy, Dermatology, autoimmunity, collagen VII, inflammation, extracellular matrix, Fc receptor, complement, granulocyte, antibody
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.
Published January 11, 2012. Keywords: Bioengineering, molecular machine, bio-nanotechnology, 5'OH DNA breaks, 5'PO4 DNA breaks, apoptosis labeling, in situ detection, vaccinia topoisomerase I, DNA breaks, green nanotechnology
JoVE Immunology and Infection
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).
Published October 18, 2010. Keywords: Immunology, Salmonella, adhesive tape, rapid detection, fresh produce, fluorescence in situ hybridization, fluorescence microscopy, flow cytometry
1Department of Pathology, University of California, Irvine (UCI)
Published October 1, 2007. Keywords: Basic Protocols, Laser Capture Microdissection, Microdissection Techniques, Leica
1Basic Research Department, The Parkinson's Institute
The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.
Published July 7, 2013. Keywords: Medicine, Stem Cell Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Molecular Biology, skin punch biopsy, skin explant culture, tissue culture, fibroblasts, primary human fibroblasts, keratinocytes, Parkinson's disease, explant, cell culture
1Biological Sciences, University of Maryland Baltimore County
Published August 10, 2013. Keywords: Anatomy, Physiology, Surgery, Tissue Engineering, Nose, Olfactory Mucosa, Olfactory Receptor Neurons, Vomeronasal Organ, skull bone removal, nasal cavity, olfactory epithelium, olfactory turbinate, respiratory epithelium, vomeronasal organ, histochemistry, mouse, animal model