JoVE: Journal of Visualized Experiments

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.

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Sun, 03 Aug 2008 14:45:00 EST

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl₂) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl₂ as cellular MRI contrast agent to determine the in vitro viability of hESC.

Major Components of the Light Microscope

Wed, 30 Jul 2008 18:18:00 EST

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

AC Electrokinetic Phenomena Generated by Microelectrode Structures

Mon, 28 Jul 2008 20:28:00 EST

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

Culture of myeloid dendritic cells from bone marrow precursors

Fri, 25 Jul 2008 18:13:00 EST

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Wed, 23 Jul 2008 22:33:00 EST

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Neutrophil Isolation Protocol

Wed, 23 Jul 2008 22:05:00 EST

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

Tue, 22 Jul 2008 00:31:00 EST

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Fri, 18 Jul 2008 23:30:00 EST

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Wed, 16 Jul 2008 17:18:00 EST

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Interview: Protein Folding and Studies of Neurodegenerative Diseases

Wed, 16 Jul 2008 16:44:00 EST

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

Single Cell Electroporation in vivo within the Intact Developing Brain

Fri, 11 Jul 2008 18:45:00 EST

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Wed, 09 Jul 2008 16:32:00 EST

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Staining Proteins in Gels

Tue, 08 Jul 2008 02:03:00 EST

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Wed, 02 Jul 2008 15:50:00 EST

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Freezing, Thawing, and Packaging Cells for Transport

Wed, 02 Jul 2008 15:30:00 EST

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Fri, 27 Jun 2008 19:56:00 EST

This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Tue, 24 Jun 2008 16:25:00 EST

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.

Counting and Determining the Viability of Cultured Cells

Mon, 23 Jun 2008 15:41:00 EST

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Immunoblot Analysis

Fri, 20 Jun 2008 14:38:00 EST

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Tue, 17 Jun 2008 17:21:00 EST

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Retrograde Labeling of Retinal Ganglion Cells by Application of Fluoro-Gold on the Surface of Superior Colliculus

Tue, 17 Jun 2008 17:19:00 EST

This video describes the method of retrograde labeling of RGC by applying fluoro-gold (FG) on the surface of superior colliculus (SC). Technique involves drilling the skull, aspirating the cortex, and applying gelatin sponge over entire dorsal surface of SC.

Operant Learning of Drosophila at the Torque Meter

Mon, 16 Jun 2008 23:19:00 EST

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

Interview: HIV-1 proviral DNA Excision Using an Evolved Recombinase

Mon, 16 Jun 2008 23:18:00 EST

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.