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Nature Protocols is an online resource for protocols, including authoritative, peer-reviewed 'Nature Protocols' and an interactive 'Protocols Network'. The two create a dynamic forum for scientists to upload and comment on protocols.

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• Using the rat forced swim test to assess antidepressant-like activity in rodents.

  Authors: David A Slattery, John F Cryan

  Nature protocols. 7(6):1009-14.

  The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and isThe forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The swim test involves the scoring of active (swimming and climbing) or passive (immobility) behavior when rodents are forced to swim in a cylinder from which there is no escape. There are two versions that are used, namely the traditional and modified FSTs, which differ in their experimental setup. For both versions, a pretest of 15 min (although a number of laboratories have used a 10-min pretest with success) is included, as this accentuates the different behaviors in the 5-min swim test following drug treatment. Reduction in passive behavior is interpreted as an antidepressant-like effect of the manipulation, provided it does not increase general locomotor activity, which could provide a false positive result in the FST.

• Genome-wide copy number analysis of single cells.

  Authors: Timour Baslan, Jude Kendall, Linda Rodgers, Hilary Cox, Mike Riggs, Asya Stepansky, Jennifer Troge, Kandasamy Ravi, Diane Esposito, B Lakshmi, Michael Wigler, Nicholas Navin, James Hicks

  Nature protocols. 7(6):1024-41.

  Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in whichCopy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ∼3 d from flow cytometry to sequence-ready DNA libraries.

• Real-time detection of acetylcholine release from the human endocrine pancreas.

  Authors: Rayner Rodriguez-Diaz, Robin Dando, Y Anthony Huang, Per-Olof Berggren, Stephen D Roper, Alejandro Caicedo

  Nature protocols. 7(6):1015-23.

  Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used asNeurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca(2+) indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca(2+) signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ∼3 h.

• Elucidating membrane structure and protein behavior using giant plasma membrane vesicles.

  Authors: Erdinc Sezgin, Hermann-Josef Kaiser, Tobias Baumgart, Petra Schwille, Kai Simons, Ilya Levental

  Nature protocols. 7(6):1042-51.

  The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context ofThe observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

• Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells.

  Authors: Erdem Karatekin, James E Rothman

  Nature protocols. 7(5):903-20.

  Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-moleculeMany biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE-reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6-8 d to complete. Analysis can take up to 2 weeks.

• Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H(2)O(2) in living Drosophila.

  Authors: Helena M Cochemé, Angela Logan, Tracy A Prime, Irina Abakumova, Caroline Quin, Stephen J McQuaker, Jigna V Patel, Ian M Fearnley, Andrew M James, Carolyn M Porteous, Robin A J Smith, Richard C Hartley, Linda Partridge, Michael P Murphy

  Nature protocols. 7(5):946-58.

  The role of hydrogen peroxide (H(2)O(2)) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H(2)O(2) in vivo. Here we describe a method forThe role of hydrogen peroxide (H(2)O(2)) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H(2)O(2) in vivo. Here we describe a method for assessing changes in H(2)O(2) within the mitochondrial matrix of living Drosophila. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H(2)O(2) to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H(2)O(2). To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography-tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H(2)O(2) in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H(2)O(2) production in mouse and cell culture studies.

• A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins.

  Authors: Jiajie Diao, Yuji Ishitsuka, Hanki Lee, Chirlmin Joo, Zengliu Su, Salman Syed, Yeon-Kyun Shin, Tae-Young Yoon, Taekjip Ha

  Nature protocols. 7(5):921-34.

  SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayersSNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

• A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine.

  Authors: Charlie Yu Ming Hsu, Hasan Uludağ

  Nature protocols. 7(5):935-45.

  This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25).This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.

• Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish.

  Authors: Juan Manuel González-Rosa, Nadia Mercader

  Nature protocols. 7(4):782-8.

  The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is basedThe zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than on tissue damage. We recently characterized the cellular response and regenerative capacity of the zebrafish heart after cryoinjury (CI), an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). After anesthesia, localized CI with a liquid nitrogen-cooled copper probe induced damage in 25% of the ventricle, in a procedure requiring <5 min. Here we present a detailed description of the technique, which provides a valuable system for the study of the mechanisms of heart regeneration and scar removal after MI in a versatile vertebrate model.

• Using bleach-chase to measure protein half-lives in living cells.

  Authors: Naama Geva-Zatorsky, Irina Issaeva, Avi Mayo, Ariel Cohen, Erez Dekel, Tamar Danon, Lydia Cohen, Yuvalal Liron, Uri Alon, Eran Eden

  Nature protocols. 7(4):801-11.

  Protein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern theseProtein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern these processes, but it is currently a major technical challenge. To address this, we developed 'bleach-chase', a noninvasive method for measuring the half-lives of multiple proteins at high temporal resolution in living cells. The method uses a library of annotated human reporter cell clones, each with a unique fluorescently tagged protein expressed from its native chromosomal location. In this protocol, we detail a simple procedure that bleaches the cells and uses time-lapse fluorescence microscopy and automated image analysis to systematically measure the half-life dynamics of multiple proteins. The duration of the protocol is 4-5 d. The method may be applicable to a wide range of fluorescently tagged proteins and cell lines.

• Mouse intragastric infusion (iG) model.

  Authors: Akiko Ueno, Raul Lazaro, Ping-Yen Wang, Reiichi Higashiyama, Keigo Machida, Hidekazu Tsukamoto

  Nature protocols. 7(4):771-81.

  Direct intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2-3 months) basis using a chronically implanted gastrostomy catheter andDirect intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2-3 months) basis using a chronically implanted gastrostomy catheter and a flow-through swivel system. This rodent intragastric infusion (iG) model has broad applications in research on food intake, gastrointestinal (GI) physiology, GI neuroendocrinology, drug metabolism and toxicity, obesity and liver disease. It achieves maximal control over the rate and pattern of delivery and it can be combined with normal ad libitum feeding of solid diet if so desired. It may be adopted to achieve infusion at other sites of the GI system to test the role of a bypassed GI segment in neuroendocrine physiology, and its use in genetic mouse models facilitates the genetic analysis of a central question under investigation.

• Inducing nonsense suppression by targeted pseudouridylation.

  Authors: Chao Huang, Guowei Wu, Yi-Tao Yu

  Nature protocols. 7(4):789-800.

  Isomerization from uridine to pseudouridine (pseudouridylation) is largely catalyzed by a family of small ribonucleoproteins called box H/ACA RNPs, each of which contains one unique small RNA-the boxIsomerization from uridine to pseudouridine (pseudouridylation) is largely catalyzed by a family of small ribonucleoproteins called box H/ACA RNPs, each of which contains one unique small RNA-the box H/ACA RNA. The specificity of the pseudouridylation reaction is determined by the base-pairing interactions between the guide sequence of the box H/ACA RNA and the target sequence within an RNA substrate. Thus, by creating a new box H/ACA RNA harboring an artificial guide sequence that base-pairs with the substrate sequence, one can site-specifically introduce pseudouridines into virtually any RNA (e.g., mRNA, ribosomal RNA, small nuclear RNA, telomerase RNA and so on). Pseudouridylation changes the properties of a uridine residue and is likely to alter the role of its corresponding RNA in certain cellular processes, thereby enabling basic research into the effects of RNA modifications. Here we take a TRM4 reporter gene (also known as NCL1) as an example, and we present a protocol for designing a box H/ACA RNA to site-specifically pseudouridylate TRM4 mRNA. Disease-related mutation can result in early termination of translation by creating a premature termination codon (PTC); however, pseudouridylation at the PTC can suppress this translation termination (nonsense suppression). Thus, the experimental procedures described in this protocol may provide a novel way to treat PTC-related diseases. This protocol takes 10-13 d to complete.

• Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA.

  Authors: Jeremy M Simon, Paul G Giresi, Ian J Davis, Jason D Lieb

  Nature protocols. 7(2):256-67.

  Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements in eukaryotic genomes. Historically identified by nuclease hypersensitivity, these regulatoryEviction 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 to identify these genomic regions and has proven successful in a multitude of eukaryotic cell and tissue types. Cells or dissociated tissues are cross-linked briefly with formaldehyde, lysed and sonicated. Sheared chromatin is subjected to phenol/chloroform extraction and the isolated DNA, typically encompassing 1-3% of the human genome, is purified. We provide guidelines for quantitative analysis by PCR, microarrays or next-generation sequencing. Regulatory elements enriched by FAIRE have high concordance with those identified by nuclease hypersensitivity or chromatin immunoprecipitation (ChIP), and the entire procedure can be completed in 3 d. FAIRE has low technical variability, which allows its usage in large-scale studies of chromatin from normal or diseased tissues.

• Microarray analysis of copy number variation in single cells.

  Authors: Peter Konings, Evelyne Vanneste, Sigrun Jackmaert, Michèle Ampe, Geert Verbeke, Yves Moreau, Joris Robert Vermeesch, Thierry Voet

  Nature protocols. 7(2):281-310.

  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 artificialWe 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 the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d.

• A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents.

  Authors: Jeffrey N Stirman, Matthew M Crane, Steven J Husson, Alexander Gottschalk, Hang Lu

  Nature protocols. 7(2):207-20.

  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 limitedOptogenetics 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 of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 μm at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.

• Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.

  Authors: Jiri Kalabis, Gabrielle S Wong, Maria E Vega, Mitsuteru Natsuizaka, Erle S Robertson, Meenhard Herlyn, Hiroshi Nakagawa, Anil K Rustgi

  Nature protocols. 7(2):235-46.

  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 modelThis 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 several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.

• Expression of high-affinity human antibody fragments in bacteria.

  Authors: Romain Rouet, David Lowe, Kip Dudgeon, Brendan Roome, Peter Schofield, David Langley, John Andrews, Peter Whitfeld, Lutz Jermutus, Daniel Christ

  Nature protocols. 7(2):364-73.

  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 periplasmicHere 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 strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

• Improved biocytin labeling and neuronal 3D reconstruction.

  Authors: Manuel Marx, Robert H Günter, Werner Hucko, Gabriele Radnikow, Dirk Feldmeyer

  Nature protocols. 7(2):394-407.

  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 differentIn 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 provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

• Single-tube linear DNA amplification for genome-wide studies using a few thousand cells.

  Authors: Pattabhiraman Shankaranarayanan, Marco-Antonio Mendoza-Parra, Wouter van Gool, Luisa M Trindade, Hinrich Gronemeyer

  Nature protocols. 7(2):328-38.

  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 beLinear 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 massive parallel sequencing (ChIP-seq) has been achieved for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplifies reChIP-seq experiments to monitor co-binding at chromatin target sites. The single-tube design of LinDA is ideal for handling ultrasmall amounts of DNA (<30 pg) and is compatible with automation. The actual hands-on working time is less than 6 h with one overnight reaction. The present protocol describes all materials and critical steps, and provides examples and controls for LinDA. Applications of LinDA for genome-wide analyses of biobank samples and for the study of chromatin conformation and nuclear architecture are in progress.

• Direct live monitoring of heterotypic axon-axon interactions in vitro.

  Authors: Liang Wang, Till Marquardt

  Nature protocols. 7(2):351-63.

  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 assayThis 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 somatosensory neuron axons, but the procedure should be readily adaptable to other neuron types. The protocol is based on the rapid isolation and primary culture of genetically identified motor neurons combined with straightforward vital dye labeling and culture of dorsal root ganglion sensory neurons. Subsequently, axonal interactions are directly monitored via live fluorescence microscopy, whereas axon type identities can be unambiguously delineated throughout the experiments. Through chemical compound application or by using neurons derived from genetically engineered mice, the protocol facilitates the dissection of molecular pathways driving the axonal interactions that are crucial for neural pathway and circuit assembly. The whole procedure can be completed in 3 d.

• Enriching libraries of high-aspect-ratio micro- or nanostructures by rapid, low-cost, benchtop nanofabrication.

  Authors: Philseok Kim, Wilmer E Adorno-Martinez, Mughees Khan, Joanna Aizenberg

  Nature protocols. 7(2):311-27.

  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-etchedWe 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 acts as an electrode for the electrodeposition of conductive polymers, transforming the original structure into a wide range of user-defined new designs. These include scaled replicas with sub-100-nm-level control of feature sizes and complex 3D shapes such as tapered or bent columnar structures bearing hierarchical features. Gradients of patterns and shapes on a single substrate can also be produced. This benchtop fabrication protocol allows the production of customized libraries of arrays of closed-cell or isolated HAR micro/nanostructures at a very low cost within 1 week, when starting from a silicon master that otherwise would be very expensive and slow to produce using conventional fabrication techniques.

• Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads.

  Authors: Adam B Robertson, John Arne Dahl, Rune Ougland, Arne Klungland

  Nature protocols. 7(2):340-50.

  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: T4We 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. We describe the steps necessary to purify JBP1 and modify this protein such that it can be fixed to magnetic beads. Thereafter, we detail how to use the JBP1 magnetic beads to obtain DNA that is enriched with 5-hmC. This method is likely to produce results similar to those of other 5-hmC pull-down assays; however, all necessary components for the completion of this protocol are readily available or can be easily and rapidly synthesized using basic molecular biology techniques. This protocol can be completed in less than 2 weeks and allows the user to isolate 5-hmC-containing genomic DNA that is suitable for analysis by quantitative PCR (qPCR), sequencing, microarray and other molecular biology assays.

• A pipeline for the generation of shRNA transgenic mice.

  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

  Nature protocols. 7(2):374-93.

  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 RNAsRNA 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, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.

• Single-mRNA counting using fluorescent in situ hybridization in budding yeast.

  Authors: Tatjana Trcek, Jeffrey A Chao, Daniel R Larson, Hye Yoon Park, Daniel Zenklusen, Shailesh M Shenoy, Robert H Singer

  Nature protocols. 7(2):408-19.

  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 andFluorescent 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 conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.

• High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy.

  Authors: Juan Carlos Tapia, Narayanan Kasthuri, Kenneth J Hayworth, Richard Schalek, Jeff W Lichtman, Stephen J Smith, JoAnn Buchanan

  Nature protocols. 7(2):193-206.

  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 inConventional 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). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

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