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Stork T.,CSHL Press | Bernardos R.,CSHL Press | Freeman M.R.,CSHL Press
Cold Spring Harbor Protocols | Year: 2012

Glial cells are the most abundant cell type in our brains, yet we understand very little about their development and function. An accumulating body of work over the last decade has revealed that glia are critical regulators of nervous system development, function, and health. Based on morphological and molecular criteria, glia in Drosophila melanogaster are very similar to their mammalian counterparts, suggesting that a detailed investigation of fly glia has the potential to add greatly to our understanding of fundamental aspects of glial cell biology. In this article, we provide an overview of the subtypes of glial cells found in Drosophila and discuss our current understanding of their functions, the development of a subset of well-defined glial lineages, and the molecular-genetic tools available for manipulating glial subtypes in vivo. © 2012 Cold Spring Harbor Laboratory Press.


Courty S.,CSHL Press | Dahan M.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

Single-molecule detection of quantum dot (QD)-tagged proteins located in the cytoplasm or the nucleus presents a significant challenge in live-cell imaging. First, QDs must enter the cell cytoplasm and reach their molecular target but still preserve cell integrity. Second, the fluorescence of individual QDs must be detected in a noisy environment and distinguished from the autofluorescence of intracellular compartments and organelles. Finally, molecular motion in the cytosol is likely to be threedimensional, compared to two-dimensional diffusion in the membrane. In this protocol, streptavidincoated QDs (QD-SAVs) are coupled with biotinylated proteins (ideally in a 1:1 molar ratio) in hypertonic medium. The coupled reaction product (QD-P) is then added to live cells (e.g., mammalian HeLa cells) using a cell-loading technique based on the osmotic lysis of pinocytic vesicles. The osmotic lysis of pinocytic vesicles in hypotonic solution does not alter the viability of cultured cells and does not result in lysosomal enzyme release. By comparison with other internalization techniques, such as microinjection, this method is much simpler and more reproducible because all of the cells are simultaneously loaded under the same conditions. It can provide quantitative information on the movement of intracellular biomolecules, enhancing our understanding of complex biological processes such as signal transduction, cell division, or motility. © 2013 Cold Spring Harbor Laboratory Press.


Caprara M.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

Information about the secondary structure of RNA 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. In this protocol we discuss chemical methods for RNA structure determination. These methods rely on the fact that certain reagents interact with RNA bases when they are single stranded, but do not react when the bases are involved in Watson-Crick base pairs. For example, dimethylsulfate (DMS) methylates the N1 position of adenosine, the N7 position of guanine, and the N3 position of cytosine only when these bases are in single-strand regions. Modifications of adenosine and cytosine create blocks to reverse transcriptase; accordingly, these modifications are detected as stops to primer extension. Modification of guanine does not create reverse transcriptase stops, but these modifications can be detected by cleavage of the modified RNA after borohydride reduction and aniline cleavage. Because DMS and other chemical reagents modify only single-stranded RNA, double-stranded regions are inferred by the lack of modification. © 2013 Cold Spring Harbor Laboratory Press.


West M.J.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

Stereology involves sampling structural features in sections of tissue with geometrical probes. This article discusses some practical issues that must be dealt with when getting started in stereology, including tissue preparation methods and determining how many tissue sections and probes are needed to make a stereological estimate. © 2013 Cold Spring Harbor Laboratory Press.


Hagendorf N.,CSHL Press | Conzelmann K.-K.,CSHL Press
Cold Spring Harbor Protocols | Year: 2015

This protocol describes the recovery of replication-competent rabies viruses (RV) such as SAD L16 or SAD L16-eGFP. It is suggested that at least three parallel transfection experiments are performed to increase the success rate (in three 3.5-cm2 dishes or in three wells in a six-well plate). The entire protocol takes 10 d, and successful rescue can be obtained after 6 d. An additional procedure for the recovery of G-gene-deleted viruses is also included. Please note that appropriate biosafety measures are needed. © 2015 Cold Spring Harbor Laboratory Press.


Ohki K.,CSHL Press | Reid R.C.,CSHL Press
Cold Spring Harbor Protocols | Year: 2014

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. © 2014 Cold Spring Harbor Laboratory Press.


Stuurman N.,CSHL Press | Swedlow J.R.,CSHL Press
Cold Spring Harbor Protocols | Year: 2012

The arrival of electronic photodetectors in biological microscopy has led to a revolution in the application of imaging in cell and developmental biology. The extreme photosensitivity of electronic photodetectors has enabled the routine use of multidimensional data acquisition spanning space and time and spectral range in live cell and tissue imaging. These techniques have provided key insights into the molecular and structural dynamics of living biology. However, digital photodetectors offer another advantage-they provide a linear mapping between the photon flux coming from the sample and the electronic sample they produce. Thus, an image presented as a visual representation of the sample is also a quantitative measurement of photon flux. These quantitative measurements are the basis of subsequent processing and analysis to improve signal contrast, to compare changes in the concentration of signal, and to reveal changes in cell structure and dynamics. For this reason, many laboratories and companies have committed their resources to software development, resulting in the availability of a large number of image-processing and analysis packages. In this article, we review the software tools for image data analysis that are now available and give some examples of their use in imaging experiments to reveal new insights into biological mechanisms. In our final section, we highlight some of the new directions for image analysis that are significant unmet challenges and present our own ideas for future directions. © 2012 Cold Spring Harbor Laboratory Press.


Hagendorf N.,CSHL Press | Conzelmann K.-K.,CSHL Press
Cold Spring Harbor Protocols | Year: 2015

G-deleted fluorescent rabies virus (RV) pseudotyped with RV G proteins, SAD ΔG eGFP (RV CVS-G), can be used as single-round vectors for efficient retrograde labeling of neurons. For these experiments, as well as for monosynaptic tracing, which involves pseudotyping in situ, the use of the CVS strain G is recommended because of its high tropism for neurons. Pseudotype virus stocks generated by transfection of pCAGGS-G (or in MG139-on cells) contain the G protein of the vaccine strain SAD L16, which is broader in its tropism, and infects astrocytes, glia, and oligodendrocytes. We also describe a procedure for pseudotyping with ASLV Env A, which uses a cell-line expressing a version of the EnvA protein that is incorporated efficiently into the RV envelope (EnvARGRGct). © 2015 Cold Spring Harbor Laboratory Press.


Hagendorf N.,CSHL Press | Conzelmann K.-K.,CSHL Press
Cold Spring Harbor Protocols | Year: 2015

Recombinant rabies virus (RV) vectors expressing fluorescent proteins allow staining of neurons from many mammalian species and enable the study of neuron morphology. Because viral spread occurs only between neurons that have synaptic connections, these vectors also permit transsynaptic tracing. A recently established system for restriction of transsynaptic tracing to a single transsynaptic jump, dubbed monosynaptic tracing, uses glycoprotein gene-defective, pseudotyped RV. This allows infection of defined cells and transient complementation with the glycoprotein in situ to support a single step of transsynaptic crossing to presynaptic cells. Here, we introduce protocols describing the production of RV vectors, including the recovery of recombinant RV from complementary DNA (cDNA) and virus pseudotyping in vitro. This allows retrograde staining of neurons projecting to the inoculation site. © 2015 Cold Spring Harbor Laboratory Press.


Rio D.C.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2-3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-D-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at -20°C. © 2013 Cold Spring Harbor Laboratory Press.

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