1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here's a look at what's coming up in the February 2013 Issue of Journal of Visualized Experiments (JoVE).
In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.
With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.
Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.
Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.
The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.
The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.
1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University
Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.
This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).
Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer
1Department of Medicine, Baylor College of Medicine, 2Division of Diabetes, Endocrinology & Metabolism, Diabetes & Endocrinology Research Center, Baylor College of Medicine, 3Department of Molecular & Cellular Biology, Baylor College of Medicine
We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.
Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.
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.
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.
Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).
Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.
Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.
Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)
Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.
An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.
Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.
Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein
The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.
Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans
Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.
This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.
Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes
This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.
The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies
The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.
Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.
Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.
Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.
Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets
A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.
Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas
Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.
We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.
Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.
1Department of Psychiatry, Center for the Study of Traumatic Stress, Uniformed Services University of the Health Sciences, Bethesda, Maryland, 2Department of Gene and Protein Biomarkers, GenProMarkers, Inc.
We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.
A general strategy for the development of charge-separating semiconductor nanocrystal composites deployable for solar energy production is presented. We show that assembly of donor-acceptor nanocrystal domains in a single nanoparticle geometry gives rise to a photocatalytic function, while bulk-heterojunctions of donor-acceptor nanocrystal films can be used for photovoltaic energy conversion.
We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.
1Institute for Solid State Research, IFW-Dresden, 2Institute of Metal Physics of National Academy of Sciences of Ukraine, 3Diamond Light Source LTD, 4Department of Physics, University of Johannesburg, 5CNR-SPIN, and Dipartimento di Fisica "E. R. Caianiello", Università di Salerno, 6Institute of Physics of Complex Matter, École Polytechnique Fédérale de Lausanne
The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.
We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection.
Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain
This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.
This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.
Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.
Moxi Z is the only automated cell counter that combines the Coulter Principle typically used in high-end cell counters with a patented thin-film sensor technology to allow for highly accurate (> 95%) and repeatable particle counting and sizing for a broad range of cell types - from mammalian cells to cells as small as wine yeast and more. Since today's workflows demand accurate quality control of samples, determining cell counts precisely has a significant impact on outcomes and downstream costs.
MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamAΔ41) from M. magneticum AMB-1.
Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format
Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.
The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.
This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.
We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 μm Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.
Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography
We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.