JoVE: Journal of Visualized Experiments

Methylated DNA Immunoprecipitation

Fri, 02 Jan 2009 00:00:00 EST

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Elevated Plus Maze for Mice

Mon, 22 Dec 2008 00:00:00 EST

The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Thu, 18 Dec 2008 00:00:00 EST

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

Electrochemotherapy of Tumours

Mon, 15 Dec 2008 00:00:00 EST

Electrochemotherapy is a combined use of certain chemotherapeutic drugs such as cisplatin and bleomycin and electric pulses applied to the treated tumour nodule. We present the clinical protocol of electrochemotherapy for treatmen of subcutaneous and cutaneous metastases of melanoma.

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Thu, 11 Dec 2008 00:00:00 EST

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Wed, 10 Dec 2008 00:00:00 EST

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

Microcontact Printing of Proteins for Cell Biology

Fri, 05 Dec 2008 00:00:00 EST

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

Mon, 01 Dec 2008 00:00:00 EST

A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Wed, 26 Nov 2008 00:00:00 EST

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan™ DIGE technology.

The 2008 Lindau Nobel Laureate Meeting: Robert Huber, Chemistry 1988

Tue, 25 Nov 2008 00:00:00 EST

Robert Huber, 1988 Nobel Laureate in Chemistry, looks to the rising generation of scientists for an “Einstein of biology” to solve the folding problem, one of sciences great mysteries.

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Fri, 21 Nov 2008 00:00:00 EST

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Wed, 19 Nov 2008 00:00:00 EST

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

Thu, 13 Nov 2008 00:00:00 EST

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Tue, 11 Nov 2008 00:00:00 EST

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Mon, 10 Nov 2008 00:00:00 EST

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

Wed, 05 Nov 2008 00:00:00 EST

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Mon, 03 Nov 2008 00:00:00 EST

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Preparation of Artificial Bilayers for Electrophysiology Experiments

Fri, 31 Oct 2008 00:00:00 EST

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

Wed, 29 Oct 2008 00:00:00 EST

A technique to collect and measure surgical wound biochemical mediators at specific time points.

Double Whole Mount in situ Hybridization of Early Chick Embryos

Mon, 27 Oct 2008 00:00:00 EST

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Wed, 22 Oct 2008 00:00:00 EST

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Mon, 20 Oct 2008 00:00:00 EST

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Fri, 17 Oct 2008 00:00:00 EST

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes

Thu, 16 Oct 2008 00:00:00 EST

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

Mon, 13 Oct 2008 00:00:00 EST

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Wed, 08 Oct 2008 00:00:00 EST

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Survivable Stereotaxic Surgery in Rodents

Mon, 06 Oct 2008 00:00:00 EST

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

Fri, 03 Oct 2008 00:00:00 EST

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Wed, 01 Oct 2008 00:00:00 EST

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

Mon, 29 Sep 2008 00:00:00 EST

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

Morris Water Maze Experiment

Wed, 24 Sep 2008 00:00:00 EST

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Wed, 17 Sep 2008 09:14:00 EST

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Mon, 15 Sep 2008 17:00:00 EST

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Thu, 11 Sep 2008 00:00:00 EST

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Sun, 07 Sep 2008 14:36:00 EST

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Wed, 03 Sep 2008 23:29:00 EST

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Tue, 02 Sep 2008 13:52:00 EST

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Preparation of 2-dGuo-Treated Thymus Organ Cultures

Thu, 28 Aug 2008 19:00:00 EST

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

Reaggregate Thymus Cultures

Thu, 28 Aug 2008 13:00:00 EST

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Preparation and Fractionation of Xenopus laevis Egg Extracts

Wed, 27 Aug 2008 12:38:00 EST

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus

Sat, 23 Aug 2008 13:32:00 EST

In this video, we demonstrate two microapplicator techniques used to infect of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Obtaining Eggs from Xenopus laevis Females

Wed, 20 Aug 2008 20:12:00 EST

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

Mon, 18 Aug 2008 17:08:00 EST

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

Generation of Stable Transgenic C. elegans Using Microinjection

Fri, 15 Aug 2008 12:12:00 EST

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Thu, 14 Aug 2008 00:00:00 EST

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

Proper Care and Cleaning of the Microscope

Mon, 11 Aug 2008 18:42:00 EST

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Mon, 11 Aug 2008 14:00:00 EST

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Fri, 08 Aug 2008 04:38:00 EST

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Wed, 06 Aug 2008 17:41:00 EST

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Tue, 05 Aug 2008 18:06:00 EST

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.