Cold Spring Harbor Protocols

Publications in this journal

• Article: In vitro imaging of retinal whole mounts.

  Philip R Williams, Joshua L Morgan, Daniel Kerschensteiner, Rachel O L Wong
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  ABSTRACT: Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vitro retinal explants. Retinas can be removed from the eye and kept in culture conditions for several days with limited disruption to the retinal circuit. The explanted retina is amenable to a variety of labeling techniques and provides a large, flat, unobstructed surface that is ideal for optical imaging experiments. This protocol describes procedures for mounting and imaging the isolated mouse retina. The same general procedure, with only minor modification (composition of culture medium), has been used to image retinas from a variety of vertebrates (e.g., chick, ferret, and rabbit).
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: Splinted Ligation Method to Detect Small RNAs.

  Timothy W Nilsen
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  ABSTRACT: In any cell, the number of RNA species is remarkably complex. The sizes of RNAs can vary from 20 nucleotides to several kilobases, and abundances can vary from a few to hundreds of thousands of molecules per cell. It is of obvious interest to determine the abundance and integrity of specific RNA species within these complex mixtures. This protocol describes the splinted ligation method to detect small RNAs. It relies on the ability of T4 DNA ligase to covalently join the terminal 3'-hydroxyl group of an RNA molecule to the labeled 5'-phosphate group of a DNA chain in the presence of a DNA "splint" or "bridge" oligonucleotide that is complementary to both. After ligation, the labeled small RNA, lengthened by the covalent addition of the (32)P-labeled oligonucleotide probe, is visualized by denaturing gel electrophoresis and phosphorimaging. This approach is recommended for the routine detection and quantification of specific small RNAs (e.g., microRNAs [miRNAs] and piwi-associated RNAs [piRNAs]).
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: Preparation of transgenic zebrafish embryos for imaging the developing retina.

  Patricia Jusuf, William A Harris, Lucia Poggi
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  ABSTRACT: The zebrafish retina is an ideal model system for addressing neural fate specification in vivo. As in all vertebrate species studied, the retina is composed of seven major cell types distributed in a laminated histogenetic arrangement. The major connections and final positioning of cell types are well known, allowing lineage tracing and identification of final cell outcome by location, morphology, and subsequent immunostaining. The retina is conveniently located on the outside of the fish, allowing the embryos to be mounted such that the eye is close to the coverslip. This enables the entire structure to be imaged in four dimensions (4D) within the given focusing depth constraints. When preparing cells for lineage tracing, it is very important to label isolated cells mosaically, so that they stand out in a mostly unlabeled background. This can be achieved by transplanting cells from transgenic or injected embryos into uninjected embryos, as is described in this protocol. Transgenic (wild-type or mutant) embryos expressing stable green or red fluorescent protein (GFP or RFP) are used as donors, and non-transgenics or transgenics expressing a different fluorescent label are used as hosts. Mosaic labeling can also be achieved by DNA injection.
  Cold Spring Harbor Protocols 01/2013; 2013(3).
• Article: Odor-taste learning assays in Drosophila larvae.

  Bertram Gerber, Roland Biernacki, Jeannette Thum
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  ABSTRACT: The Drosophila larva is an emerging model for studies in behavioral neurogenetics because of its simplicity in terms of cell number. Despite this simplicity, basic features of neuronal organization and key behavior faculties are shared with adult flies and with mammals. Here, we describe a Pavlovian-type learning assay in fruit fly larvae. A group of larvae is sequentially exposed to specific odors in the presence or the absence of sugar, and then tested to determine whether they prefer the odor previously experienced with the reward. The protocol uses a two-group, reciprocal training design: One group of Drosophila larvae is exposed to n-amyl acetate (AM) with a sugar reward (+), then subsequently exposed to 1-octanol (OCT) with no reward (denoted AM+/OCT). The other group receives the reciprocal training (AM/OCT+). The two groups of larvae are then tested for their choices between AM and OCT. Relatively higher preferences for AM after AM+/OCT training than after AM/OCT+ training reflect associative learning and are quantified by the learning index (LI). This method offers a robust, simple, cheap, and reasonably quick test for learning ability (an aversive version is available as well, using either high-concentration salt or quinine as punishment). With the concerted efforts of the Drosophila research community, we anticipate it will allow us to unravel the full circuitry underlying odor-taste learning on a single-cell level.
  Cold Spring Harbor Protocols 01/2013; 2013(3).
• Article: RNA structure determination using nuclease digestion.

  Timothy W Nilsen
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  ABSTRACT: Determining RNA structures (i.e., single- and double-strand regions) is often useful when assessing the potential for certain RNAs to interact with proteins or when determining whether RNAs that are dissimilar in sequence can form the same structure. A number of ribonucleases (RNases) have been used to map RNA structure, but many of these are no longer available. However, three commonly available RNA endonucleases (RNase T1, RNase I, and RNase V1) can provide a wealth of structural information. Cleavages of end-labeled RNA are initiated by one of the RNases (H2O is used for mock-treated controls), terminated with aurintricarboxylic acid (a potent RNase inhibitor), and detected by electrophoresis on denaturing polyacrylamide gels. Because there are enzymes that can cleave only when the RNA is single stranded (e.g., RNase T1) or double stranded (e.g., RNase V1), it is possible to do parallel analyses.
  Cold Spring Harbor Protocols 01/2013; 2013(4).
• Article: Clonal analysis of olfaction in Drosophila: generation of flies with mosaic labeling.

  Aaron Ostrovsky, Sebastian Cachero, Gregory Jefferis
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  ABSTRACT: Clonal analysis with the MARCM (mosaic analysis with a repressible cell marker) system can be used for studying cell lineage, development, and anatomy in the Drosophila olfactory system and other parts of the fly brain. This protocol gives a method for generating flies with mosaic labeling. It describes how to establish a mating cage for MARCM in PNs (projection neurons) of the fly antennal lobe and then select appropriate flies for dissection and staining using immunohistochemistry. The protocol can be adapted to determine the birth order of neuroblast lineages or individual cells. Alternatively, it can be used to dissect a complicated Gal4 line into its component neuroblast lineages to help elucidate projection patterns and connectivity. Collecting newly hatched larvae during a short time window allows for precise control of the stage during development at which the heat shock is applied.
  Cold Spring Harbor Protocols 01/2013; 2013(4).
• Article: How to make calcium-sensitive minielectrodes.

  Roger C Thomas, Donald M Bers
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  ABSTRACT: In this protocol we describe how to make and use minielectrodes for measuring [Ca(2+)] in small volumes of solution. The minielectrodes are ∼2 mm in diameter and have sufficiently low resistances to be used with a standard pH meter. They are made by dipping polyethylene or borosilicate glass tubes (∼5 cm long) in a membrane solution. Although the chemicals used to make these Ca(2+)-sensitive minielectrodes are expensive, they can be used to make hundreds of electrodes, each with a useful life of several months.
  Cold Spring Harbor Protocols 01/2013; 2013(4).
• Article: The primer extension assay.

  Michael F Carey, Craig L Peterson, Stephen T Smale
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  ABSTRACT: In the primer extension assay, the transcription start site for a gene is determined experimentally by identifying the 5' end of the encoded messenger RNA (mRNA). The protocol begins with a primer, usually a synthetic oligonucleotide of about 20 residues, that is complementary to an mRNA sequence ∼50-150 nucleotides downstream of the anticipated 5' end. The primer is 5'-end-labeled using [γ-(32)P]ATP and T4 polynucleotide kinase and is annealed to the specific mRNA molecules within an RNA sample. Reverse transcriptase (RT), deoxyribonucleoside triphosphates, and appropriate buffer components are added to the primer-mRNA hybrids to catalyze elongation of the primer to the 5' end of the mRNA. The resulting radiolabeled complementary DNA (cDNA) products are analyzed by denaturing polyacrylamide gel electrophoresis, followed by autoradiography. The sizes of the bands detected on the gel, as compared to an adjacent sequencing ladder or molecular weight standards, provide a measure of the distance from the 5' end of the synthetic oligonucleotide to the beginning of the mRNA transcripts. In theory, the 3' end of the cDNA will coincide with the 5' end of the mRNA. Thus, the size of the radiolabeled cDNAs should represent the distance from the labeled 5' end of the primer to the 5' end of the mRNA (i.e., the 3' end of the cDNA). If the labeled cDNA products are within the resolution range of the gel, the transcription start site can be determined with an accuracy of plus or minus one nucleotide.
  Cold Spring Harbor Protocols 01/2013; 2013(2).
• Article: Isolation of mouse mammary organoids for long-term time-lapse imaging.

  Andrew J Ewald
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  ABSTRACT: Many epithelial tissues develop deep within the animal and are inaccessible to high-resolution optical imaging with visible wavelengths. Protocols for culturing whole epithelial organs have existed since the 1950s, but the use of three-dimensional (3D) organotypic cultures of epithelial fragments has advanced dramatically in recent years. Here we describe the practical details involved in isolating mammary epithelial tissue and culturing organoids embedded within 3D gels. Mammary glands are mechanically disrupted and enzymatically digested, and the epithelial cell fragments are separated from stromal cells by differential centrifugation. The organoids are cultured in BD Matrigel in the absence or presence of growth factor.
  Cold Spring Harbor Protocols 01/2013; 2013(2).
• Article: Gel purification of RNA.

  Timothy W Nilsen
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  ABSTRACT: For many applications, including size selection of RNAs and purification of in vitro transcription products, it is necessary to purify RNAs on a denaturing gel. This procedure describes how to purify transcripts that have been synthesized in vitro. It is useful for labeled or unlabeled RNAs when sufficient mass is present. It can also be used to isolate small RNAs. In general, RNA purification by denaturing gel electrophoresis is practical only when the size of the desired RNA is 600 nucleotides or less.
  Cold Spring Harbor Protocols 01/2013; 2013(2).
• Article: Analyzing Ca2+ dynamics in intact epithelial cells using spatially limited flash photolysis.

  Janos Almassy, David I Yule
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  ABSTRACT: The production of saliva by parotid acinar cells is stimulated by Ca(2+) activation of Cl(-) and K(+) channels located in the apical plasma membrane of these polarized cells. Here we describe a paradigm for the focal photorelease of either Ca(2+) or an inositol 1,4,5 trisphosphate (InsP(3)) analog. The protocol is designed to be useful for investigating subcellular Ca(2+) dynamics in polarized cells with minimal experimental intervention. Parotid acinar cells are loaded with cell-permeable versions of the caged precursors (NP-EGTA-AM or Ci-InsP(3)/PM). Photolysis is accomplished using a spatially limited, focused diode laser, but the experiment can be readily modified to whole-field photolysis using a xenon flash lamp.
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: In vivo imaging of zebrafish retina.

  Philip R Williams, Joshua L Morgan, Daniel Kerschensteiner, Rachel O L Wong
  [show abstract] [hide abstract]
  ABSTRACT: Neuronal circuits of the vertebrate retina are organized into stereotyped laminae. This orderly arrangement makes the retina an ideal model system for imaging studies aimed at understanding how circuits assemble during development. In particular, live-cell imaging techniques are readily applied to the developing retina to monitor dynamic changes over time in cell structure and connectivity. Such imaging studies have collectively revealed novel strategies by which retinal neurons contact their presynaptic and postsynaptic partners to establish synaptic connections. We describe here the procedures developed in our laboratory for confocal and multiphoton live-cell imaging of the developing retina using in vivo preparations. Zebrafish larvae are an ideal specimen for in vivo imaging experiments as they can be made to remain transparent throughout development. Isolated retinal cells can be readily labeled by DNA injection into the one-cell staged embryo, or via transplantation of fluorescently labeled cells from stable transgenics.
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: Reconstitution of holo-aequorin with apoaequorin mRNA and coelenterazine in zebrafish embryos.

  Ching Man Chan, Andrew L Miller, Sarah E Webb
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  ABSTRACT: When holo-aequorin is injected into zebrafish embryos at the one-cell stage, it is normally depleted by ∼24 h post-fertilization (hpf). In order to acquire Ca(2+) signaling information from embryos older than 24 hpf, we have developed a protocol to express apoaequorin transiently in embryos, after which we reconstitute active holo-aequorin in vivo by introducing the cofactor coelenterazine into the developing embryo. This protocol describes the preparation of apoaequorin mRNA, followed by microinjection into embryos and incubation with coelenterazine to reconstitute holo-aequorin.
  Cold Spring Harbor Protocols 01/2013; 2013(5).
• Article: Introduction of Aequorin into Zebrafish Embryos for Recording Ca2+ Signaling during the First 48 h of Development.

  Sarah E Webb, Ching Man Chan, Andrew L Miller
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  ABSTRACT: Ca(2+) signals, whether transient pulses, propagating waves, or long-duration, steady gradients, are generally considered to play an important role in the pattern-forming events that occur during vertebrate development. One vertebrate that has long been a favorite of embryologists because of its ex utero development and the optical clarity of its embryos is the zebrafish, Danio rerio. Using the bioluminescent Ca(2+) reporter aequorin, distinct Ca(2+) signals have been reported for at least the first 48 h of zebrafish development, with signals becoming progressively more complex as the embryo develops. Here we provide a general introduction to aequorin and its use in monitoring Ca(2+) signals and discuss methods for introducing aequorin into zebrafish embryos.
  Cold Spring Harbor Protocols 01/2013; 2013(5).
• Article: Quantitative imaging of morphogen gradients in Drosophila imaginal discs.

  Anna Kicheva, Laurent Holtzer, Ortrud Wartlick, Thomas Schmidt, Marcos González-Gaitán
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  ABSTRACT: Cells at different positions in a developing tissue receive different concentrations of signaling molecules, called morphogens, and this influences their cell fate. Morphogen concentration gradients have been proposed to control patterning as well as growth in many developing tissues. Some outstanding questions about tissue patterning by morphogen gradients are the following: What are the mechanisms that regulate gradient formation and shape? Is the positional information encoded in the gradient sufficiently precise to determine the positions of target gene domain boundaries? What are the temporal dynamics of gradients and how do they relate to patterning and growth? These questions are inherently quantitative in nature and addressing them requires measuring morphogen concentrations in cells, levels of downstream signaling activity, and kinetics of morphogen transport. Here we first present methods for quantifying morphogen gradient shape in which the measurements can be calibrated to reflect actual morphogen concentrations. We then discuss using fluorescence recovery after photobleaching to study the kinetics of morphogen transport at the tissue level. Finally, we present particle tracking as a method to study morphogen intracellular trafficking.
  Cold Spring Harbor Protocols 01/2013; 2013(5).
• Article: Imaging calcium waves in cerebellar bergmann glia.

  Michael Beierlein
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  ABSTRACT: This protocol describes methods for recording synaptically evoked Ca(2+) waves from individual Bergmann glia (BG) in slices of cerebellar cortex. Unlike protoplasmic, star-shaped astrocytes, whose thin processes pose a serious challenge to stable Ca(2+) measurements, BG are large radial cells, with several main processes that run over distances of several hundred micrometers toward the pia and ensheathe thousands of parallel fiber (PF) synapses. Stimulation of PF synapses with brief bursts can trigger long-lasting Ca(2+) responses in BG processes, which can be reliably recorded using a cooled charge-coupled device (CCD) camera. This protocol was developed to enable measurements of Ca(2+) waves in individual BG loaded with a high-affinity Ca(2+) indicator such as Fura-2 for up to 2 h. Because BG recorded in slices rarely display spontaneous (i.e., tetrodotoxin [TTX]-sensitive) or intrinsic Ca(2+) transients, Ca(2+) waves can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Fluorescence measurements obtained using CCD technology offer a straightforward means of characterizing the mechanisms and potential functional consequences of widespread and long-lasting, store-mediated Ca(2+) increases in astrocytes.
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: Reverse Genetics by Loss-of-Function Mosaic Analysis in Drosophila.

  Chih-Fei Kao, Tzumin Lee
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  ABSTRACT: Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. One major application of MARCM, as described here, is to study cell-autonomous function(s) of a gene within single cells or a group of cells in otherwise unperturbed organisms. A mutation of interest distal to one FRT site is put in trans to a tubP-GAL80-containing chromosome arm that carries the same FRT. The resulting MARCM clones, which are negative for tubP-GAL80 and thus specifically marked, will become homozygous for the mutation in otherwise heterozygous organisms. By including a UAS-transgene, one can perform rescue experiments in the mutant MARCM clones. Conversely, if the mutation is placed on the same chromosome arm as tubP-GAL80, MARCM-labeled cells will be homozygous wild-type and may lie adjacent to sister cells that are homozygous mutant. This variant, called reverse MARCM, allows one to determine non-cell-autonomous effects of a mutation.
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: High-speed two-photon imaging.

  Gaddum Duemani Reddy, Peter Saggau
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  ABSTRACT: The small size of neuronal dendrites and spines combined with the high speed of neurophysiological signals, such as transients in membrane potential or ion concentration, necessitates that any functional study of these structures uses recording methods with both high spatial and high temporal resolutions. In this regard, conventional two-photon microscopy, in combination with fluorescent indicators sensitive to physiological parameters, has proved to be only a partial solution by providing near-diffraction-limited spatial resolution even when imaging structures deep inside light-scattering tissue. This is because the relatively slow beam-scanning methods used in most conventional two-photon microscopes severely limit the extent to which functional data can be recorded. Here, we detail developments to create high-speed two-photon imaging systems that overcome this limitation and discuss important considerations that must be taken into account when attempting to construct such systems.
  Cold Spring Harbor Protocols 01/2013; 2013(1).
• Article: DNase I Footprinting.

  Michael F Carey, Craig L Peterson, Stephen T Smale
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  ABSTRACT: DNase I footprinting has found a wide following for both identifying and characterizing DNA-protein interactions, particularly because of its simplicity. The concept is that a partial digestion by DNase I of a uniquely (32)P-end-labeled fragment will generate a ladder of fragments, whose mobilities on a denaturing acrylamide gel and whose positions in a subsequent autoradiograph will represent the distance from the end label to the points of cleavage. Bound protein prevents binding of DNase I in and around its binding site and thus generates a "footprint" in the cleavage ladder. The distance from the end label to the edges of the footprint represents the position of the protein-binding site on the DNA fragment. The position of the binding site can be determined by electrophoresing a DNA sequencing ladder alongside the footprint. DNase I cannot bind directly adjacent to a DNA-bound protein because of steric hindrance. Hence, the footprint gives a broad indication of the binding site, generally 8-10 base pairs (bp) larger than the site itself.
  Cold Spring Harbor Protocols 01/2013; 2013(5).
• Article: Inducing RNAi in Drosophila Cells by Transfection with dsRNA.

  Rui Zhou, Stephanie Mohr, Gregory J Hannon, Norbert Perrimon
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  ABSTRACT: In Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN's Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.
  Cold Spring Harbor Protocols 01/2013; 2013(5).