JoVE: Journal of Visualized Experiments

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

Thu, 02 Jul 2009 00:00:00 EST

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

The 2009 Lindau Nobel Laureate Meeting: Aaron Ciechanover, Chemistry 2004

Wed, 01 Jul 2009 00:00:00 EST

Aaron Ciechanover was born in Haifa, Israel in October 1947. Ciechanover shared the Nobel Prize in Chemistry in 2004 with Avram Hershko and Irwin Rose for their discovery of ubiquitin-mediated protein degradation.

Making Gynogenetic Diploid Zebrafish by Early Pressure

Tue, 30 Jun 2009 00:00:00 EST

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Focal Ca²⁺ Transient Detection in Smooth Muscle

Mon, 29 Jun 2009 00:00:00 EST

Details methods for high-resolution Ca²⁺ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

An interview with Nobel laureate Roy Glauber, Physics 2005

Fri, 26 Jun 2009 00:00:00 EST

The field of quantum optics rests on the work of Roy Glauber, who helped elucidate the nature of light as both particles and waves. In this candid interview, the Nobel prize-winning physicist shares his thoughts about this strange and unintuitive behavior of light, balancing fatherhood with an academic career, and working at Los Alamos National Laboratory, where he shockingly learned that he was helping to build The Bomb.

Measuring Replicative Life Span in the Budding Yeast

Thu, 25 Jun 2009 00:00:00 EST

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Implantation of Engineered Tissue in the Rat Heart

Wed, 24 Jun 2009 00:00:00 EST

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Tue, 23 Jun 2009 00:00:00 EST

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Small Volume (1-3L) Filtration of Coastal Seawater Samples

Fri, 19 Jun 2009 00:00:00 EST

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

Large Volume (20L+) Filtration of Coastal Seawater Samples

Thu, 18 Jun 2009 00:00:00 EST

This video documents large volume (≥20 L) filtration of microbial biomass, ranging between 0.22μm and 2.7μm in diameter, from the water column.

Seawater Sampling and Collection

Wed, 17 Jun 2009 19:00:00 EST

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.

Primary Culture of Adult Rat Heart Myocytes

Tue, 16 Jun 2009 00:00:00 EST

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Monitoring Plant Hormones During Stress Responses

Mon, 15 Jun 2009 00:00:00 EST

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Fri, 12 Jun 2009 00:00:00 EST

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Catheterization of Intestinal Loops in Ruminants

Thu, 11 Jun 2009 00:00:00 EST

We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

Wed, 10 Jun 2009 00:00:00 EST

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Tue, 09 Jun 2009 00:00:00 EST

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Mon, 08 Jun 2009 00:00:00 EST

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Dendra2 Photoswitching through the Mammary Imaging Window

Fri, 05 Jun 2009 00:00:00 EST

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Thu, 04 Jun 2009 00:00:00 EST

In this video we present the microfluidic probe¹ (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

A Tactile Automated Passive-Finger Stimulator (TAPS)

Wed, 03 Jun 2009 00:00:00 EST

We describe a computer-controlled device for investigating the sense of touch: the Tactile Automated Passive-finger Stimulator (TAPS). We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Tue, 02 Jun 2009 00:00:00 EST

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Mon, 01 Jun 2009 00:00:00 EST

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

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

Fri, 29 May 2009 00:00:00 EST

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Homarus Americanus Stomatogastric Nervous System Dissection

Thu, 28 May 2009 00:00:00 EST

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

The Gateway to the Brain: Dissecting the Primate Eye

Wed, 27 May 2009 00:00:00 EST

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Tue, 26 May 2009 00:00:00 EST

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Tue, 26 May 2009 00:00:00 EST

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

Fri, 22 May 2009 00:00:00 EST

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

Electrophysiological Recording in the Drosophila Embryo

Thu, 21 May 2009 00:00:00 EST

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Wed, 20 May 2009 00:00:00 EST

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Dissection of Saccharomyces Cerevisiae Asci

Tue, 19 May 2009 00:00:00 EST

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Mon, 18 May 2009 23:00:00 EST

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

Fri, 15 May 2009 00:00:00 EST

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

Thu, 14 May 2009 00:00:00 EST

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space¹.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Wed, 13 May 2009 00:00:00 EST

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Measuring Caenorhabditis elegans Life Span on Solid Media

Tue, 12 May 2009 00:00:00 EST

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Homemade Site Directed Mutagenesis of Whole Plasmids

Mon, 11 May 2009 00:00:00 EST

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Fri, 08 May 2009 00:00:00 EST

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Thu, 07 May 2009 00:00:00 EST

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

Wed, 06 May 2009 00:00:00 EST

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Tue, 05 May 2009 00:00:00 EST

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Fabrication of Amperometric Electrodes

Mon, 04 May 2009 00:00:00 EST

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabrication of Myogenic Engineered Tissue Constructs

Fri, 01 May 2009 00:00:00 EST

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block^[1].

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Wed, 29 Apr 2009 00:00:00 EST

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Sun, 26 Apr 2009 23:27:00 EST

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Fri, 24 Apr 2009 00:00:00 EST

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.

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

Wed, 22 Apr 2009 00:00:00 EST

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Mon, 20 Apr 2009 00:00:00 EST

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

Fri, 17 Apr 2009 00:00:00 EST

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.