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Nature Protocols
Expression of high-affinity human antibody fragments in bacteria
Thu, 02/02/2012 - 00:00Expression of high-affinity human antibody fragments in bacteria
Nature Protocols 7, 364 (2012). doi:10.1038/nprot.2011.448
Authors: Romain Rouet, David Lowe, Kip Dudgeon, Brendan Roome, Peter Schofield, David Langley, John Andrews, Peter Whitfeld, Lutz Jermutus & Daniel Christ
Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification
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A pipeline for the generation of shRNA transgenic mice
Thu, 02/02/2012 - 00:00A pipeline for the generation of shRNA transgenic mice
Nature Protocols 7, 374 (2012). doi:10.1038/nprot.2011.446
Authors: Lukas E Dow, Prem K Premsrirut, Johannes Zuber, Christof Fellmann, Katherine McJunkin, Cornelius Miething, Youngkyu Park, Ross A Dickins, Gregory J Hannon & Scott W Lowe
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches—inducible and reversible gene silencing. However,
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Improved biocytin labeling and neuronal 3D reconstruction
Thu, 02/02/2012 - 00:00Improved biocytin labeling and neuronal 3D reconstruction
Nature Protocols 7, 394 (2012). doi:10.1038/nprot.2011.449
Authors: Manuel Marx, Robert H Günter, Werner Hucko, Gabriele Radnikow & Dirk Feldmeyer
In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We
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Single-mRNA counting using fluorescent in situ hybridization in budding yeast
Thu, 02/02/2012 - 00:00Single-mRNA counting using fluorescent in situ hybridization in budding yeast
Nature Protocols 7, 408 (2012). doi:10.1038/nprot.2011.451
Authors: Tatjana Trcek, Jeffrey A Chao, Daniel R Larson, Hye Yoon Park, Daniel Zenklusen, Shailesh M Shenoy & Robert H Singer
Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each
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Enriching libraries of high-aspect-ratio micro- or nanostructures by rapid, low-cost, benchtop nanofabrication
Thu, 01/26/2012 - 00:00Enriching libraries of high-aspect-ratio micro- or nanostructures by rapid, low-cost, benchtop nanofabrication
Nature Protocols 7, 311 (2012). doi:10.1038/nprot.2012.003
Authors: Philseok Kim, Wilmer E Adorno-Martinez, Mughees Khan & Joanna Aizenberg
We provide a protocol for transforming the structure of an array of high-aspect-ratio (HAR) micro/nanostructures into various new geometries. Polymeric HAR arrays are replicated from a Bosch-etched silicon master pattern by soft lithography. By using various conditions, the original pattern is coated with metal, which
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Single-tube linear DNA amplification for genome-wide studies using a few thousand cells
Thu, 01/26/2012 - 00:00Single-tube linear DNA amplification for genome-wide studies using a few thousand cells
Nature Protocols 7, 328 (2012). doi:10.1038/nprot.2011.447
Authors: Pattabhiraman Shankaranarayanan, Marco-Antonio Mendoza-Parra, Wouter van Gool, Luisa M Trindade & Hinrich Gronemeyer
Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled to
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Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads
Thu, 01/26/2012 - 00:00Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads
Nature Protocols 7, 340 (2012). doi:10.1038/nprot.2011.443
Authors: Adam B Robertson, John Arne Dahl, Rune Ougland & Arne Klungland
We describe a method for the efficient and selective identification of DNA containing the 5-hydroxymethylcytosine (5-hmC) modification. This protocol takes advantage of two proteins: T4 β-glucosyltransferase (β-gt), which converts 5-hmC to β-glucosyl-5-hmC (β-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to β-glu-5-hmC.
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Direct live monitoring of heterotypic axon-axon interactions in vitro
Thu, 01/26/2012 - 00:00Direct live monitoring of heterotypic axon-axon interactions in vitro
Nature Protocols 7, 351 (2012). doi:10.1038/nprot.2011.442
Authors: Liang Wang & Till Marquardt
This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and
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In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix
Thu, 01/19/2012 - 00:00In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix
Nature Protocols 7, 247 (2012). doi:10.1038/nprot.2011.440
Authors: Jieun Kim, Nicole Rubin, Ying Huang, Tai-Lan Tuan & Ching-Ling Lien
We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in
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Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA
Thu, 01/19/2012 - 00:00Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA
Nature Protocols 7, 256 (2012). doi:10.1038/nprot.2011.444
Authors: Jeremy M Simon, Paul G Giresi, Ian J Davis & Jason D Lieb
Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements in eukaryotic genomes. Historically identified by nuclease hypersensitivity, these regulatory elements are typically bound by transcription factors or other regulatory proteins. FAIRE (formaldehyde-assisted isolation of regulatory elements) is an alternative approach
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Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue
Thu, 01/19/2012 - 00:00Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue
Nature Protocols 7, 268 (2012). doi:10.1038/nprot.2011.445
Authors: Vanessa Gil & José Antonio del Río
This protocol uses rat tail–derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat
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Microarray analysis of copy number variation in single cells
Thu, 01/19/2012 - 00:00Microarray analysis of copy number variation in single cells
Nature Protocols 7, 281 (2012). doi:10.1038/nprot.2011.426
Authors: Peter Konings, Evelyne Vanneste, Sigrun Jackmaert, Michèle Ampe, Geert Verbeke, Yves Moreau, Joris Robert Vermeesch & Thierry Voet
We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000–bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on
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High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy
Thu, 01/12/2012 - 00:00High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy
Nature Protocols 7, 193 (2012). doi:10.1038/nprot.2011.439
Authors: Juan Carlos Tapia, Narayanan Kasthuri, Kenneth J Hayworth, Richard Schalek, Jeff W Lichtman, Stephen J Smith & JoAnn Buchanan
Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM).
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A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents
Thu, 01/12/2012 - 00:00A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents
Nature Protocols 7, 207 (2012). doi:10.1038/nprot.2011.433
Authors: Jeffrey N Stirman, Matthew M Crane, Steven J Husson, Alexander Gottschalk & Hang Lu
Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting
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Intravital multiphoton imaging of immune responses in the mouse ear skin
Thu, 01/12/2012 - 00:00Intravital multiphoton imaging of immune responses in the mouse ear skin
Nature Protocols 7, 221 (2012). doi:10.1038/nprot.2011.438
Authors: Jackson LiangYao Li, Chi Ching Goh, Jo L Keeble, Jim S Qin, Ben Roediger, Rohit Jain, Yilin Wang, Weng Keong Chew, Wolfgang Weninger & Lai Guan Ng
Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin,
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Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture
Thu, 01/12/2012 - 00:00Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture
Nature Protocols 7, 235 (2012). doi:10.1038/nprot.2011.437
Authors: Jiri Kalabis, Gabrielle S Wong, Maria E Vega, Mitsuteru Natsuizaka, Erle S Robertson, Meenhard Herlyn, Hiroshi Nakagawa & Anil K Rustgi
This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating
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Quantitative monitoring of mouse lung tumors by magnetic resonance imaging
Thu, 01/05/2012 - 00:00Quantitative monitoring of mouse lung tumors by magnetic resonance imaging
Nature Protocols 7, 128 (2012). doi:10.1038/nprot.2011.424
Authors: Alexander Sasha Krupnick, Vanessa K Tidwell, John A Engelbach, Vamsi V Alli, Arye Nehorai, Ming You, Haris G Vikis, Andrew E Gelman, Daniel Kreisel & Joel R Garbow
Primary lung cancer remains the leading cause of cancer-related death in the Western world, and the lung is a common site for recurrence of extrathoracic malignancies. Small-animal (rodent) models of cancer can have a very valuable role in the development of improved therapeutic strategies. However,
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Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp
Thu, 01/05/2012 - 00:00Tracking mechanics and volume of globular cells with atomic force microscopy using a constant-height clamp
Nature Protocols 7, 143 (2012). doi:10.1038/nprot.2011.434
Authors: Martin P Stewart, Yusuke Toyoda, Anthony A Hyman & Daniel J Müller
To understand the role of physical forces at a cellular level, it is necessary to track mechanical properties during cellular processes. Here we present a protocol that uses flat atomic force microscopy (AFM) cantilevers clamped at constant height, and light microscopy to measure the resistance
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High-throughput ballistic injection nanorheology to measure cell mechanics
Thu, 01/05/2012 - 00:00High-throughput ballistic injection nanorheology to measure cell mechanics
Nature Protocols 7, 155 (2012). doi:10.1038/nprot.2011.436
Authors: Pei-Hsun Wu, Christopher M Hale, Wei-Chiang Chen, Jerry S H Lee, Yiider Tseng & Denis Wirtz
High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories of
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A transcription activator-like effector toolbox for genome engineering
Thu, 01/05/2012 - 00:00A transcription activator-like effector toolbox for genome engineering
Nature Protocols 7, 171 (2012). doi:10.1038/nprot.2011.431
Authors: Neville E Sanjana, Le Cong, Yang Zhou, Margaret M Cunniff, Guoping Feng & Feng Zhang
Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34–amino acid repeat modules that can be rearranged according to a simple cipher to target new
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