JoVE: Journal of Visualized Experiments

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Wed, 01 Sep 2010 17:00:00 EST

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using ³¹P NMR Spectroscopy

Tue, 31 Aug 2010 17:00:00 EST

Langendorff-mode isolated heart perfusion, in conjunction with ³¹P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Intraperitoneal Injection into Adult Zebrafish

Mon, 30 Aug 2010 17:00:00 EST

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 μl NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Direct Tracheal Instillation of Solutes into Mouse Lung

Sun, 29 Aug 2010 17:00:00 EST

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Sat, 28 Aug 2010 17:00:00 EST

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Fri, 27 Aug 2010 17:30:00 EST

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

Thu, 26 Aug 2010 17:00:00 EST

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Wed, 25 Aug 2010 17:30:00 EST

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Experimental Metastasis Assay

Tue, 24 Aug 2010 17:10:00 EST

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Generation of Transgenic C. elegans by Biolistic Transformation

Mon, 23 Aug 2010 17:00:00 EST

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

RhoC GTPase Activation Assay

Sun, 22 Aug 2010 17:00:00 EST

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Sat, 21 Aug 2010 17:00:00 EST

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Novel Apparatus and Method for Drug Reinforcement

Fri, 20 Aug 2010 17:00:00 EST

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Simultaneous fMRI and Electrophysiology in the Rodent Brain

Thu, 19 Aug 2010 17:00:00 EST

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Wed, 18 Aug 2010 17:00:00 EST

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Tue, 17 Aug 2010 17:00:00 EST

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

相比肿瘤细胞皮下注射或腹腔注射的异种移植模型,同位动物模型的主要优势在于它能更好的表现人类癌症,所以也能使我们更好的了解人类癌症及其对治疗的反应.

Tue, 17 Aug 2010 17:00:00 EST

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Mon, 16 Aug 2010 17:00:00 EST

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Eye Movement Monitoring of Memory

Sun, 15 Aug 2010 17:00:00 EST

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Sat, 14 Aug 2010 17:00:00 EST

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Fri, 13 Aug 2010 17:00:00 EST

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Thu, 12 Aug 2010 17:30:00 EST

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Wed, 11 Aug 2010 17:00:00 EST

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tue, 10 Aug 2010 17:00:00 EST

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Mon, 09 Aug 2010 17:00:00 EST

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

Murine echocardiography and ultrasound imaging

Sun, 08 Aug 2010 19:00:00 EST

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation

Sat, 07 Aug 2010 17:00:00 EST

Microscopic analysis of γH2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced γH2AX formation within the nucleus.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

Fri, 06 Aug 2010 17:30:00 EST

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

Transurethral Induction of Mouse Urinary Tract Infection

Thu, 05 Aug 2010 17:30:00 EST

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

Wed, 04 Aug 2010 17:32:00 EST

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

Generation of Recombinant Influenza Virus from Plasmid DNA

Tue, 03 Aug 2010 17:10:00 EST

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

DNA Stable-Isotope Probing (DNA-SIP)

Mon, 02 Aug 2010 17:25:00 EST

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.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Sun, 01 Aug 2010 18:00:00 EST

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.

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Sat, 31 Jul 2010 17:00:00 EST

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Fri, 30 Jul 2010 17:30:00 EST

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Thu, 29 Jul 2010 17:00:00 EST

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

Wed, 28 Jul 2010 17:00:00 EST

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Tue, 27 Jul 2010 17:00:00 EST

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Primer-Free Aptamer Selection Using A Random DNA Library

Mon, 26 Jul 2010 17:00:00 EST

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Labeling and Imaging Cells in the Zebrafish Hindbrain

Sun, 25 Jul 2010 17:00:00 EST

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Basics of Multivariate Analysis in Neuroimaging Data

Sat, 24 Jul 2010 17:00:00 EST

The current article describes the basics of multivariate analysis and contrasts it to the more commonly used voxel-wise univariate analysis. Both types of analysis are applied to a clinical-neuroscience data set. Supplementary split-half simulations show better replication of the multivariate results in independent data sets.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Fri, 23 Jul 2010 17:30:00 EST

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Thu, 22 Jul 2010 17:00:00 EST

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Wed, 21 Jul 2010 17:24:00 EST

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Tue, 20 Jul 2010 17:45:00 EST

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Mon, 19 Jul 2010 17:00:00 EST

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Dissection of Oenocytes from Adult Drosophila melanogaster

Sun, 18 Jul 2010 17:00:00 EST

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Murine Bioluminescent Hepatic Tumour Model

Sat, 17 Jul 2010 17:00:00 EST

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Fri, 16 Jul 2010 17:00:00 EST

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Thu, 15 Jul 2010 17:00:00 EST

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.