JoVE: Journal of Visualized Experiments

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Tue, 09 Mar 2010 17:05:05 EST

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Mon, 08 Mar 2010 16:41:00 EST

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Murine Model of CD40-activation of B cells

Fri, 05 Mar 2010 17:30:09 EST

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Dissection of Organs from the Adult Zebrafish

Thu, 04 Mar 2010 16:45:00 EST

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Wed, 03 Mar 2010 17:30:00 EST

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Tue, 02 Mar 2010 17:57:00 EST

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Multielectrode Array Recordings of the Vomeronasal Epithelium

Mon, 01 Mar 2010 16:25:00 EST

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Fri, 26 Feb 2010 16:45:00 EST

In this article we will describe the procedure for measuring diffusion coefficients using multi-photon fluorescence recovery after photobleaching. We will begin by aligning the laser along the optical path to the sample and determining the proper experimental parameters, then continue generating and finally fitting fluorescence recovery curves.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Thu, 25 Feb 2010 16:50:00 EST

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Wed, 24 Feb 2010 17:00:00 EST

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Tue, 23 Feb 2010 17:20:00 EST

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Mon, 22 Feb 2010 17:00:00 EST

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Fri, 19 Feb 2010 17:10:00 EST

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Fluorescence Activated Cell Sorting of Plant Protoplasts

Thu, 18 Feb 2010 17:30:00 EST

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Wed, 17 Feb 2010 14:25:00 EST

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Tue, 16 Feb 2010 15:05:00 EST

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

Mon, 15 Feb 2010 17:00:00 EST

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Fri, 12 Feb 2010 18:30:00 EST

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Thu, 11 Feb 2010 04:52:00 EST

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

The 2009 Lindau Nobel Laureate Meeting: Martin Chalfie, Chemistry 2008

Wed, 10 Feb 2010 18:45:00 EST

American Biologist Martin Chalfie shared the 2008 Nobel Prize in Chemistry with Roger Tsien and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Chalfie subcloned the coding sequence of GFP and expressed it in both E. coli and C. elegans, demonstrating for the first time that no other factor was required for GFP luminescence.

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Tue, 09 Feb 2010 17:00:00 EST

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Mon, 08 Feb 2010 17:30:00 EST

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Depletion of Specific Cell Populations by Complement Depletion

Fri, 05 Feb 2010 14:00:00 EST

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

Thu, 04 Feb 2010 00:00:00 EST

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Wed, 03 Feb 2010 16:25:00 EST

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Using the optokinetic response to study visual function of zebrafish

Tue, 02 Feb 2010 18:45:00 EST

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults^1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Mon, 01 Feb 2010 16:25:00 EST

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Fri, 29 Jan 2010 00:00:00 EST

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Isolation of Mouse Peritoneal Cavity Cells

Thu, 28 Jan 2010 16:45:00 EST

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)

Wed, 27 Jan 2010 16:25:00 EST

Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Tue, 26 Jan 2010 17:00:00 EST

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Mon, 25 Jan 2010 16:30:00 EST

This video describes a right hemihepatectomy with intrahepatic transection of the right hemipedicle leaving the hepatoduodenal ligament completely untouched.

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Fri, 22 Jan 2010 16:52:00 EST

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Thu, 21 Jan 2010 00:00:00 EST

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Erratum: Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

Wed, 20 Jan 2010 23:57:00 EST

A correction to author names has been made for article Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease

Wed, 20 Jan 2010 18:15:00 EST

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Tue, 19 Jan 2010 17:00:00 EST

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Spinal Cord Electrophysiology

Mon, 18 Jan 2010 16:00:00 EST

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Fri, 15 Jan 2010 00:00:00 EST

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Visualizing RNA Localization in Xenopus Oocytes

Thu, 14 Jan 2010 17:00:00 EST

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

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Thu, 14 Jan 2010 00:00:00 EST

The 2009 Lindau Nobel Laureate Meeting: Roger Y. Tsien, Chemistry 2008

Wed, 13 Jan 2010 16:01:00 EST

American biochemist Roger Tsien shared the 2008 Nobel Prize in Chemistry with Martin Chalfie and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Tsien dramatically improved the wild-type GFP resulting in increased fluorescence, increased photostability, and a shift in the major excitation peak to 488 nm (matching FITC).

A Protocol for the Production of KLRG1 Tetramer

Tue, 12 Jan 2010 16:01:00 EST

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

In vivo-like Organotypic Murine Retinal Wholemount Culture

Mon, 11 Jan 2010 17:30:00 EST

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Fri, 08 Jan 2010 17:00:00 EST

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Thu, 07 Jan 2010 00:00:00 EST

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Wed, 06 Jan 2010 17:10:00 EST

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Tue, 05 Jan 2010 20:00:00 EST

We will show how to record flash responses from single mouse cones using a suction electrode.

Presynaptically Silent Synapses Studied with Light Microscopy

Mon, 04 Jan 2010 17:00:00 EST

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Thu, 31 Dec 2009 14:00:00 EST

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.