Cold Spring Harbor Protocols

Publisher: Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press


Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is a definitive, interactive source of new and classic research techniques. The database is fully searchable by keyword and subject, and it has many novel features - such as discussion forums and personal folders - made possible by online publication. Its coverage includes cell and molecular biology, genetics, bioinformatics, protein science, and imaging. Protocols are presented step-by-step and edited in the style that has made Molecular Cloning, Antibodies, Cells and many other CSH manuals essential to the work of scientists worldwide. Protocols will be continuously expanded, updated, and annotated by the originators and users of the techniques. CSH Protocols - continuing Cold Spring Harbor Laboratory's 60-year tradition as a source of trusted techniques.

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    Cold Spring Harbour Protocols website
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    CSH protocols, Protocols, CSH protocols, CSH protocols online
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    Document, Periodical, Internet resource
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    Internet Resource, Computer File, Journal / Magazine / Newspaper

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Cold Spring Harbor Laboratory Press

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    • Publisher last contacted on 15/07/2013
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Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescence lifetime imaging (FLIM) is a powerful imaging mode that can be combined with confocal imaging. Changes in the fluorescence decay time of a donor in an intramolecular Förster resonance energy transfer (FRET)-based biosensor provide intrinsic quantitative data. Here, we describe a protocol using both the Ca2+ sensor TN-XL, which uses troponin C, as the Ca2+-sensing unit, and the FLIM technology based on time-correlated single-photon counting.
    Cold Spring Harbor Protocols 12/2014;
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    ABSTRACT: This protocol describes a method for the in vivo measurement of steroid hormones in brain circuits of the zebra finch. A guide cannula is surgically implanted into the skull, microdilysate is collected through a microdialysis probe that is inserted into the cannula, and steroid concentrations in the microdialysate are determined using the enzyme-linked immunosorbent assay (ELISA). In some cases, the steroids measured are derived locally (e.g., neural estrogens in males), whereas in other cases, the steroids measured reflect systemic circulating levels and/or central conversion (e.g., the primary androgen testosterone and the primary glucocorticoid corticosterone). A reverse-microdialysis ("retrodialysis") method that can be used to deliver pharmacological agents into the brain to influence local steroid neurochemistry as well as behavior is also discussed.
    Cold Spring Harbor Protocols 10/2014;
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    ABSTRACT: Songbirds are capable of learning their vocalizations by copying a singing adult. This vocal learning ability requires juveniles to hear and memorize the sound of the adult song, and later to imitate it through a process involving sensorimotor integration. Vocal learning is a trait that songbirds share with humans, where it forms the basis of spoken language acquisition, with other avian groups (parrots and hummingbirds), and with a few other mammals (cetaceans, bats). It is however absent in traditional model organisms such as rodents and nonhuman primates. Zebra finches, a songbird species from Australia, are popular pets and are easy to breed. They also sing a relatively simple and stereotyped song that is amenable to quantitative analysis. Zebra finches have thus emerged as a choice model organism for investigating the neurobiological basis of vocal learning. A number of tools and methodologies have been developed to characterize the bioacoustics properties of their song, analyze the degree of accurate copying during vocal learning, map the brain circuits that control singing and song learning, and investigate the physiology of these circuits. Such studies have led to a large base of knowledge on song production and learning, and their underlying neural substrate. Several molecular resources have recently become available, including brain cDNA/EST databases, microarrays, BAC libraries, a molecular brain atlas, a complete genome assembly, and the ability to perform transgenesis. The recent availability of many other avian genomes provides unique opportunities for comparative analysis in the search for features unique to vocal learning organisms.
    Cold Spring Harbor Protocols 10/2014;
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    ABSTRACT: The zebra finch is an important model for investigating the neural mechanisms that underlie vocal production and learning. Previous anatomical and gene expression studies have identified an interconnected set of brain areas in this organism that are important for singing. To advance our understanding of how these various brain areas act together to learn and produce a highly stereotyped song, it is necessary to record the activity of individual neurons during singing. Here, we present a protocol for recording single-unit activity in freely moving zebra finches during singing using a miniature, motorized microdrive. It includes procedures for both the microdrive implant surgery and the electrophysiological recordings. There are several advantages of this technique: (1) high-impedance electrodes can be used in the microdrive to obtain well-isolated single units; (2) a motorized microdrive is used to remotely control the electrode position, allowing neurons to be isolated without handling the bird, and (3) a lateral positioner is used to move electrodes into fresh tissue before each penetration, allowing recordings from well-isolated neurons over the course of several weeks. We also describe the application of the antidromic stimulation and the spike collision test to identify neurons based on the axonal projection patterns.
    Cold Spring Harbor Protocols 10/2014;
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    ABSTRACT: In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-µm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.
    Cold Spring Harbor Protocols 10/2014;
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    ABSTRACT: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a major messenger for Ca(2+) mobilization in cells. NAADP-binding proteins are highly selective and have a strong affinity for NAADP. This is the basis of the radioreceptor binding assay, which is used to measure NAADP levels in cells and tissues and to identify cellular stimuli that use NAADP as an intracellular messenger. In the radioreceptor binding assay, radiolabeled NAADP ([(32)P]NAADP) competes with endogenous NAADP present in samples for binding to their receptors. Here, we describe the synthesis of [(32)P]NAADP for use in the radioreceptor binding assay.
    Cold Spring Harbor Protocols 09/2014; 2014(9):pdb.prot076919.
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    ABSTRACT: When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.
    Cold Spring Harbor Protocols 09/2014; 2014(9):pdb.top074567.
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    ABSTRACT: Flies with mutations in Atg7 or Atg8a are homozygous viable and develop to adulthood, whereas mutations in other autophagy genes, including Atg1, are homozygous lethal. Clonal analysis has been instrumental in examining the role and regulation of lethal Atg genes in many aspects of Drosophila development and survival. The generation of homozygous mutant clones in an otherwise heterozygous mutant background is possible in mitotically active tissues, and is highly beneficial in that the control cells and experimental cells are subjected to the same developmental and nutritional cues allowing for a side-by-side comparison. Several methods are now available to examine the contribution of lethal autophagy genes during Drosophila oogenesis. Here we describe how to generate a homozygous mutant germline using the FLP-DFS (dominant female sterile) technique, how to generate somatic clones, and how to induce targeted gene knockdown in the germline using RNAi.
    Cold Spring Harbor Protocols 09/2014; 2014(9):pdb.prot080358.
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    ABSTRACT: Two-photon imaging of calcium-sensitive dyes in vivo has become a common tool used by neuroscientists, largely because of the development of bolus loading techniques, which can label every neuron in a local circuit with calcium-sensitive dye. Like multielectrode recordings, two-photon imaging paired with bolus loading provides a method for monitoring many neurons at once, but, in addition, it provides a means for determining the precise location of every neuron. Thus, it is an ideal method for studying the fine-scale functional architecture of the cortex and guiding the experimenter to individual neurons that can be targeted for further anatomical study. Two-photon calcium imaging enables study of the fine structure of functional maps in the visual cortex in cats and rodents. In mice, it can allow the characterization of specific cell types when paired with transgenic or retrograde labeling.
    Cold Spring Harbor Protocols 09/2014; 2014(4).
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    ABSTRACT: The apparent Ca(2+) affinity of the isoforms of the sarco/endoplasmic reticulum Ca(2+) ATPase SERCA2 is controlled primarily by two proteins, phospholamban (PLB) and sarcolipin (SLN). The rate of ATP-driven Ca(2+) uptake into sarcoplasmic reticulum (SR)-derived vesicles can be monitored by a technique in which the net uptake of (45)Ca(2+) in the form of an intravesicular calcium oxalate precipitate is recorded. Here, we present details of a modification of such a protocol for determining the apparent Ca(2+) affinity of the Ca(2+) pump, and its control by various regulators, in crude homogenates of mouse heart.
    Cold Spring Harbor Protocols 08/2014; 2014(8):pdb.prot076893.
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    ABSTRACT: This protocol describes how to generate and analyze products and intermediates in a pre-mRNA splicing reaction. The reaction relies on the use of labeled, capped, synthetic pre-mRNAs, prepared by in vitro transcription, and Drosophila Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1-2 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea-acrylamide gel. Visualization of the splicing reactions will reveal the pre-mRNA, the spliced mRNA, and the intermediates generated by the first step of splicing. For inefficient reactions, a more sensitive detection method, such as RNase protection, primer extension, or RT-PCR (reverse transcription-polymerase chain reaction), may be required.
    Cold Spring Harbor Protocols 08/2014; 2014(8):pdb.prot080853.
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    ABSTRACT: Tumor-suppressor genes are critical regulators of growth and functioning of cells, whose loss of function contributes to tumorigenesis. Accordingly, analyses of the consequences of their loss of function in genetically engineered mouse models have provided important insights into mechanisms of human cancer, as well as resources for preclinical analyses and biomarker discovery. Nowadays, most investigations of genetically engineered mouse models of tumor-suppressor function use conditional or inducible alleles, which enable analyses in specific cancer (tissue) types and overcome the consequences of embryonic lethality of germline loss of function of essential tumor-suppressor genes. However, historically, analyses of genetically engineered mouse models based on germline loss of function of tumor-suppressor genes were very important as these early studies established the principle that loss of function could be studied in mouse cancer models and also enabled analyses of these essential genes in an organismal context. Although the cancer phenotypes of these early germline models did not always recapitulate the expected phenotypes in human cancer, these models provided the essential foundation for the more sophisticated conditional and inducible models that are currently in use. Here, we describe these "first-generation" germline models of loss of function models, focusing on the important lessons learned from their analyses, which helped in the design and analyses of "next-generation" genetically engineered mouse models.
    Cold Spring Harbor Protocols 08/2014; 2014(8):pdb.top069773.
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    ABSTRACT: Selective expression of opsins in genetically defined neurons makes it possible to control a subset of neurons without affecting nearby cells and processes in the intact brain, but light must still be delivered to the target brain structure. Light scattering limits the delivery of light from the surface of the brain. For this reason, we have developed a fiber-optic-based optical neural interface (ONI), which allows optical access to any brain structure in freely moving mammals. The ONI system is constructed by modifying the small animal cannula system from PlasticsOne. The system for bilateral stimulation consists of a bilateral cannula guide that has been stereotactically implanted over the target brain region, a screw cap for securing the optical fiber to the animal's head, a fiber guard modified from the internal cannula adapter, and a bare fiber whose length is customized based on the depth of the target region. For unilateral stimulation, a single-fiber system can be constructed using unilateral cannula parts from PlasticsOne. We describe here the preparation of the bilateral ONI system and its use in optical stimulation of the mouse or rat brain. Delivery of opsin-expressing virus and implantation of the ONI may be conducted in the same surgical session; alternatively, with a transgenic animal no opsin virus is delivered during the surgery. Similar procedures are useful for deep or superficial injections (even for neocortical targets, although in some cases surface light-emitting diodes or cortex-apposed fibers can be used for the most superficial cortical targets).
    Cold Spring Harbor Protocols 08/2014; 2014(8):pdb.prot083337.
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    ABSTRACT: This protocol describes an extremely sensitive procedure for detecting the presence of known or unknown RNAs in a complex mixture. A selectively enriched population of RNAs is subjected to 3'-end labeling with [(32)P]pCp, and labeled products are separated from unincorporated label. The labeled RNAs are hybridized to sequence-specific complementary oligodeoxynucleotides, treated with RNase H (which cleaves RNA in an RNA-DNA hybrid) and the products analyzed by electrophoresis through denaturing polyacrylamide gels with the appropriate controls. If the RNA of interest was present and hybridized to its complementary oligonucleotide, its digestion with RNase H will result in a shift in its mobility through the gel or, if the RNA was fully degraded, its band will not appear. If the RNA of interest is not cleaved in the presence of any known complementary oligodeoxynucleotides, then its position in the gel will remain unaltered. This result may suggest the presence of a new or unknown RNA that may be identified using a variety of cloning techniques or by direct chemical sequencing methods.
    Cold Spring Harbor Protocols 07/2014; 2014(7):pdb.prot080796.
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    ABSTRACT: Conventional techniques used to measure Ca(2+) binding are too slow to determine accurately the fast binding kinetics of most molecules such as Ca(2+)-binding proteins (CBPs). We have developed an ultra-fast in vitro technique for measuring the Ca(2+)-binding properties of CBPs following flash photolysis of caged Ca(2+). Although the details of the setup, the mathematics, and the analysis involved in this technique have been published elsewhere, many of the practical details regarding the actual measurements have, until now, only been described minimally. Here, we present a protocol to gather the data necessary to determine the kinetic properties of a caged-Ca(2+) compound and a CBP.
    Cold Spring Harbor Protocols 07/2014; 2014(7):pdb.prot073296.
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    ABSTRACT: We describe a method for labeling presynaptic terminals in mammalian brain slices by focal application of calcium indicators conjugated with acetoxymethyl (AM) esters. A solution of membrane-permeant, AM-conjugated calcium indicator is focally applied to the transverse cerebellar brain slice and allowed to equilibrate throughout the parallel fiber tract. Fibers are then stimulated with an extracellular electrode and fluorescence transients are measured from a location several hundred micrometers from the loading site using a photodiode or photomultiplier tube. Considerations for selecting an appropriate indicator and determining the optimum loading conditions are discussed.
    Cold Spring Harbor Protocols 07/2014; 2014(7):pdb.prot081828.
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    ABSTRACT: This protocol describes the detection of small RNAs (∼10-200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Because high-percentage polyacrylamide gels are required to separate RNAs in this size range, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a (32)P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea-polyacrylamide gel electrophoresis.
    Cold Spring Harbor Protocols 07/2014; 2014(7):pdb.prot080838.