Springer protocols feed by Neuroscience 
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.In Vivo Dual Luciferase Reporter Assay with Chick Neural Tube In Ovo Electroporation System
Luciferase reporter systems are widely employed to provide a quantitative readout of gene expression for studies of transcriptional regulation, translation efficiency, and cell signaling. The most common application of luciferase involves transient transfections into cells in vitro or in vivo. In both cases, the normal variability inherent in transfection approaches can introduce significant errors into the data that makes comparison between separate experiments problematic. The dual luciferase reporter assay system (DLR, Promega, WI, USA) is designed to control for this technical issue by using a co-transfection approach ...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Chromatin Immunoprecipitation Assay of Brain Tissues Using Percoll Gradient-Purified Nuclei
Protein–DNA interactions are critical to maintain genome stability, DNA replication, chromosome segregation and to regulate gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique to study these interactions within living neurons and nervous tissue. In particular, ChIP analysis of chromatin in which protein–DNA interactions are first fixed in situ provides a valuable approach to identify specific transcription factor–DNA interactions and their regulation in the developing nervous system. Here we describe a procedure utilizing Percoll gradient purification of nuclei from fresh br...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
β-Galactosidase Staining of LacZ Fusion Proteins in Whole Tissue Preparations
The lacZ gene product, β-galactosidase, has classically been used as a reporter of gene expression. β-Galactosidase activity can be detected using a chromogenic substrate, X-gal, which leaves an intense blue precipitate when cleaved by the enzyme. Insertion of the lacZ coding DNA targeted into a specific gene creates a β-galactosidase-tagged fusion protein that is expressed under the endogenous promoter. Analysis of the hybrid protein takes advantage of the chromogenic detection system, as the distribution and relative abundance of the expressed protein can be efficiently visualized. (Source: Springer protoc...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Immunofluorescence Staining with Frozen Mouse or Chick Embryonic Tissue Sections
Immunofluorescence (IF), a form of immunohistochemistry (IHC) with specific applications, is commonly used for both basic research and clinical studies, including diagnostics, and involves visualizing the cellular distribution of target molecules (e.g., proteins, DNA, and small molecules) using a microscope capable of exciting and detecting fluorochrome compounds that emit light at specific, largely nonoverlapping wavelengths. The procedure for carrying out IF varies according to the tissue type and methods for processing and preparing tissue (e.g., fixative used to preserve tissue morphology and antigenicity). The protoco...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
General Introduction to In Situ Hybridization Protocol Using Nonradioactively Labeled Probes to Detect mRNAs on Tissue Sections
In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (In Situ) or in the entire tissue (whole mount ISH). Localization of endogenous transcripts is a desirable approach for confirming expression patterns. This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. However, RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissu...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
In Utero Electroporation in Mice
In utero electroporation has been extensively used to study a variety of developmental questions in the developing brain. This protocol aims to provide the basic knowledge for a beginner to get familiar with the technique. Basically, by electroporating a DNA construct into a subpopulation of progenitor cells in the ventricular zone of embryonic brain, the progenitor cells carrying the DNA will undergo neurogenesis, migration, and final differentiation to become mature neurons positioned in distinct cortical layers according to their birth date. In addition, by controlling the direction of electroporation, a specific cortic...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Gene Transfer in Developing Chick Embryos: In Ovo Electroporation
In ovo electroporation is a popular technique to study gene function during development. This technique enables precise temporal and spatial genetic manipulation with the added advantages of being quick and inexpensive. In this chapter the transient transfection of a construct into the neural tube of a chicken embryo via in ovo electroporation is described. Modifications of this basic technique and methods to analyze the resulting electroporated embryos such as qPCR and microarray are also discussed. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
In Ovo Electroporation in Embryonic Chick Spinal Cords
The developing spinal cord is a well-established model system widely used to study the signaling pathways and genetic programs that control neuronal/glial differentiation and neural circuit assembly. This is largely due to the relatively simple organization (compared to other CNS regions) and experimental accessibility of the neural tube, particularly in the chick embryo. In vivo transfection of cells within the developing chick neural tube using in ovo electroporation has emerged as a rapid and powerful experimental technique in that (1) transfected factors can be functionally tested in a spatially and temporally controll...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Lentiviral Vector Production, Titration, and Transduction of Primary Neurons
Lentiviral vectors have become very useful tools for transgene delivery. Based on their ability to transduce both dividing and nondividing cells and to produce long-term transgene expression, lentiviruses have found numerous applications in the biomedical sciences, including developmental neuroscience. This protocol describes how to prepare lentiviral vectors by calcium phosphate transfection and to concentrate viral particles by ultracentrifugation. Functional vector titers can then be determined by methods such as fluorescence-activated cell sorting or immunostaining. Effective titers in the range of 108–109 infect...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
The Gene-Gun Approach for Transfection and Labeling of Cells in Brain Slices
Biolistic transfection and diolistic labeling are techniques in which subcellular-sized particles, coated with DNA and lipophilic dyes, respectively, are propelled into cells. The gene-gun approach is particularly applicable for use on ex vivo organized tissue such as brain slices, where cells are not accessible for transfection with methods used in dissociated cell preparations. This simple and rapid method results in targeting of individual cells in a Golgi-like manner, allowing investigating structural and functional aspects of neuronal development. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
DNA Transfection: Calcium Phosphate Method
The calcium phosphate transfection is a widely used method for introducing foreign DNA plasmids into cells. Mechanisms underlying this transfection method are not yet defined; however, DNA–calcium phosphate precipitates are internalized by the cells and DNA is efficiently expressed in almost all cell types. The cost-efficiency and simplicity of this method allows for use in primary neuronal cultures, despite issues of neurotoxicity. Here, we describe an optimized calcium phosphate transfection method for the delivery of DNA plasmid into primary dissociated neuronal cultures. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Isolation and Culture of Schwann Cells
Primarily cultured Schwann cells are essential for the investigation of molecular mechanisms regulating proliferation, survival, differentiation, and myelination of Schwann cell and for the development of efficient transplantation for regeneration of injured spinal cord or peripheral nervous system. Here we describe a basic protocol for isolation and purification of primary Schwann cell from neonatal rat or mouse and discuss some modifications adapted to the culturing from adult nerves and optional methods for Schwann cell enrichment. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Culturing Oligodendrocyte Lineage Cells from Neonatal Rats
The use of enriched oligodendrocyte lineage cell cultures has yielded insight into functions of these cells and regulatory mechanisms. This chapter details methods that result in such cultures. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Culturing Astrocytes from Postnatal Rats
The use of cultures has informed us of functions and regulation of astrocytes that were previously unknown. This chapter details the methods that result in such cultures. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
Preparation of Primary Cultured Dopaminergic Neurons from Mouse Brain
Dopaminergic neurons are involved in a variety of normal brain functions; degenerations of these neurons cause diseases in human. Investigation of how dopaminergic neurons respond to extracellular signals and molecular mechanisms regulating dopaminergic neuron survival and death often requires reliable, reproducible, and high-quality primary cultures of dopaminergic neurons. Here, we described methods to dissect and culture these neurons from embryonic mesencephalon of mouse brain. We utilize coverslips made from Aclar film to maximize the number of surviving dopaminergic neuron in the culture and immunocytochemistry of ty...
Source: Springer protocols feed by Neuroscience - May 27, 2013 Category: Neuroscience Source Type: news
