JoVE General
Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks
1Molecular and Microbiology Department and Center for the Study of Genomics in Liver Diseases, George Mason University, 2Translational Research Institute, Inova Health System, 3Research Center for Medical Genetics RAMS
Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.
JoVE Editorial
May 2012: This Month in JoVE
Here are some highlights from the May 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE Immunology and Infection
Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock
1Agriculture and Agri-Food Canada, Lethbridge Research Centre, 2Department of Bioscience and Biotechnology, Dalian University of Technology, 3Agriculture Centre, Alberta Agriculture and Rural Development
Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure. Once infectious agent is confirmed non-viable, mature compost can be spread as fertilizer.
JoVE Neuroscience
SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval
1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University
We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.
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 General
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
Max-Planck Institute for Evolutionary Anthropology, Leipzig
We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.
JoVE General
Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue
We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.
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
Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection
Department of Microbiology, Immunology and Pathology, Colorado State University
Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.
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 Neuroscience
Extinction Training During the Reconsolidation Window Prevents Recovery of Fear
1Departments of Psychiatry and Neuroscience, and Friedman Brain Institute, Mt. Sinai School of Medicine, 2Department of Psychology, New York University, 3Department of Psychology and Center for Neural Science, New York University
Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).
JoVE General
Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies
1Department of Pathology, University of Wisconsin - Milwaukee, 2BioInnovation LLC
Practicing of fine needle aspiration biopsies (FNAB) by trainees is relatively challenging, due to the lack of an easily available, appropriate lesion. Preparation of an AV phantom lesion for practicing the FNAB procedure and mastering proficiency is relatively easy.
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
A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
1Department of Microbiology, Medical School, University of Minnesota, 2Department of Veterinary and Biomedical Sciences, University of Minnesota
A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.
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 General
Chromatin Isolation by RNA Purification (ChIRP)
ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.
JoVE Clinical and Translational Medicine
Murine Fetal Echocardiography
Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.
JoVE Application Notes
The Waters Xevo™ TQ-S Equipped With New StepWave™ and ScanWave™ Technology - ADVERTISEMENT
Pharmaceutical Business Operations, Waters Corporation
JoVE General
Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation
1Department of Chemistry, Tunghai University, 2Department of Biochemistry, University of California, Riverside
MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.
JoVE General
DNA Stable-Isotope Probing (DNA-SIP)
Department of Biology, University of Waterloo
DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.
JoVE Clinical and Translational Medicine
Staining Protocols for Human Pancreatic Islets
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida
This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.
JoVE Immunology and Infection
Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines
U.S. Army Medical Research Institute of Infectious Diseases
Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.
JoVE General
Single Oocyte Bisulfite Mutagenesis
1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute
Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.
JoVE General
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.
JoVE Clinical and Translational Medicine
Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain
1Department of Surgery, University of Texas Medical Branch, 2Department of Neuroscience and Cell Biology, University of Texas Medical Branch
A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.
JoVE Neuroscience
Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging
Department of Neurological Surgery, University of California, San Francisco - UCSF
Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.
JoVE General
Isolation and Characterization of RNA-Containing Exosomes
This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.
JoVE Immunology and Infection
Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles
Institute of Biochemical Engineering, Technische Universität Braunschweig
A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.
JoVE Neuroscience
Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion
1Centre for Neuroscience, University of Alberta, 2Neuroscience Program, University of Illinois, Urbana-Champaign, 3Center for Cognitive Neuroscience, Duke University, 4Psychology Department, Neuroscience Program, & Beckman Institute, University of Illinois, Urbana-Champaign
We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.
JoVE Neuroscience
Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection
1Department of Psychiatry, University of Alberta, Edmonton, 2Psychology Department, University of Illinois, Urbana-Champaign, 3Neuroscience Program, University of Illinois, Urbana-Champaign, 4Beckman Institute for Advanced Science & Technology, University of Illinois, Urbana-Champaign
We present a protocol that allows investigation of the neural correlates of recollecting emotional autobiographical memories, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.
JoVE General
Retrieval of Mouse Oocytes
Dept. of Biological Chemistry, University of California, Irvine (UCI)
This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.
JoVE Clinical and Translational Medicine
Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis
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.
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 Clinical and Translational Medicine
Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats
Medicine/Nephrology, Indiana University School of Medicine
A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.
JoVE Clinical and Translational Medicine
Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease
Department of Neurology, Vanderbilt University
The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.
JoVE Neuroscience
Wholemount Immunohistochemistry for Revealing Complex Brain Topography
1Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, 2Department of Cell Biology and Anatomy and the Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary
Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.
JoVE Neuroscience
Behavioral Assessment of Manual Dexterity in Non-Human Primates
Department of Medicine, University of Fribourg
As manual dexterity is a prerogative mainly of primates, behavioral tasks have been developed in macaque monkeys. Four reach and grasp prehension tasks, measuring hand manipulation ability and force, allow to establish functional recovery after a lesion of the central nervous system and to test the effect of a treatment.
JoVE General
Brain Banking: Making the Most of your Research Specimens
1Department of Physiology, University of Montreal, 2School of Optometry, University of Montreal
Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.
JoVE Immunology and Infection
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center
We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.
JoVE General
Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies
Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.
JoVE General
Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
JoVE Neuroscience
Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells
Department of Cell and Developmental Biology, Vanderbilt University
This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.
JoVE Neuroscience
Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice
1Department of Cell and Developmental Biology, Vanderbilt University, 2Department of Neurology, Vanderbilt University
This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.
JoVE Bioengineering
Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts
Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.
JoVE General
Seawater Sampling and Collection
Department of Microbiology and Immunology, University of British Columbia - UBC
This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.
JoVE General
A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat
Department of Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge
In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.
JoVE General
Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos
Unitat Biologia Cellular (Facultat de Biociències), Universitat Autonoma de Barcelona
In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.
JoVE General
Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes
We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.
JoVE General
A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types
1Department of Applied Mathematics & Statistics, Stony Brook University, 2Computational Biology and Bioinformatics, Cold Spring Harbor Laboratory, 3Department of Molecular and Cell Biology, University of Texas at Dallas
Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.
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.
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