Springer protocols feed by Neuroscience 
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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.Assaying AMPA Receptor Oligomerization
Functional AMPA receptors (AMPARs) are tetrameric complexes formed by four identical (homomeric) or similar (heteromeric) subunits. Variations in the number and composition of AMPARs on the plasma membrane impact synaptic strength, neurodevelopment, and brain disorders. While the mechanisms mediating oligomerization of AMPARs are not well understood, they form the template for defining the number and preferential assembly of AMPARs. In this chapter we describe the application of two methods, blue-native PAGE (BN-PAGE) and fluorescence-detection size-exclusion chromatography (FSEC), to delineate the oligomeric state of AMPA...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Quantum Dot-Based In Situ Hybridization and Immunohistochemistry to Detect mRNA and Protein at Subcellular Levels, Comparison with Studies Using Electron Microscopy
Semiconductor nanocrystals (Quantum dots, Qdots) have advantages of narrow, symmetric emission spectra with multiple resolvable colors that can be excited simultaneously using a single excitation wavelength. Therefore, different sized Qdots can serve the simultaneous detection of mRNA and protein. The procedure of in situ hybridization (ISH) and immunohistochemistry (IHC) using confocal laser scanning microscopy (CLSM) and Qdots is simpler than combined ISH and IHC using electron microscopy. This method is applicable to the three-dimensional detection of several molecules including mRNA and proteins. (Source: Springer prot...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Multiplex Fluorescent RNA In Situ Hybridization Via RNAscope
Multiplex fluorescent in situ hybridization is a useful tool for neurobiology applications. We have developed a novel RNA ISH technology, RNAscope, with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. Here, we present a detailed protocol of Multiplex Fluorescent RNAscope Assay that permits visualization of three target genes simultaneously on fresh frozen brain tissue sections. The step-by-step protocol describes tissue preparation, fixation, pretreatment, probe hybridization, signal amplific...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Application of Alkaline Phosphatase-Mediated Azo Dye Staining for Dual Fluorescent In Situ Hybridization in Zebrafish
We report a dual fluorescent in situ hybridization (FISH) method for direct comparison of cellular distributions of different gene transcripts in the embryonic zebrafish brain and other tissues. After simultaneous hybridization of two differently labeled antisense RNA probes, the different hapten labels are visualized by peroxidase (POD)-mediated deposition of fluorochrome-labeled tyramides and alkaline phosphatase-based Fast Blue or Fast Red chromogenic staining, respectively. Since chromogenic Fast Blue and Fast Red precipitates display red-fluorescent emission, multiplexed visualization of different transcripts is possi...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Subcellular Transcript Localization in Drosophila Embryos and Tissues Visualized by Multiplex-FISH
Determining the spatiotemporal expression dynamics of a gene, or the subcellular localization properties of its encoded RNA, is often a key first step toward elucidating its function. Fluorescent in situ hybridization (FISH) represents the gold standard method for visualizing RNA expression and subcellular localization features in distinct cells, tissue specimens, and whole-mount organisms. This chapter describes a high-resolution FISH protocol for the detection of coding or noncoding RNA expression and localization dynamics in embryos and tissues of the fruit fly, Drosophila melanogaster. Variations of the protocol are pr...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Fluorescent In Situ Hybridization Detection of microRNAs in Newt Tissue Cryosections
Even though numerous protocols exist using in situ hybridization techniques to identify RNA targets within whole mount samples or tissue sections, a methodological finesse is still required in order to obtain specific labelling, free of high non-specific background staining. One parameter that is paramount to the specificity of staining is the in situ probe design. Large RNA molecules can be probed with long DNA oligonucleotide sequences that bind with high specificity to their target RNA, which have high melting temperatures and facilitate high stringency washing. However, small RNA targets, like 20–24 bp long micro...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Combining Whole-Mount In Situ Hybridization with Neuronal Tracing and Immunohistochemistry
Despite the presence of several markers to study the expression of genes (eGFP, LacZ), in situ hybridization remains one of the most powerful techniques to analyze gene expression. While this allows cellular identification of the expression of a single gene and, using fluorescent in situ hybridization two or occasionally more genes, it is often necessary to combine this technology with assays of neuronal projection/morphology, protein expression using antibody staining, and histology for cytological details. Since each task has certain levels of false negatives, combining them in a single preparation can compromise further...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Fluorescent In Situ Hybridization in Primary Hippocampal Neurons to Detect Localized mRNAs
In neurons, local translation of mRNAs contributes to axon outgrowth and activity-dependent synaptic plasticity. The identification and visualization of individual localized mRNAs is critical for understanding these processes. Here, we describe a sensitive fluorescence in situ hybridization (FISH) method that provides high-resolution information on mRNA localization using in vitro cultured rat hippocampal neurons. The method employs digoxigenin (DIG)-labeled RNA probes and fluorescent tyramide signal amplification for detection of mRNAs. It enables not only the visualization but also quantification of mRNA granules, and ch...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Analysis of Gene Expression in Neurons and Synapses by Multi-color In Situ Hybridization
In situ hybridization is a powerful and highly specific method to reveal cell and tissue specific gene expression. It is especially critical for neuroscience applications where the enormous heterogeneity of cells as well as tissue composition present a significant methodological and conceptual challenge. Here we present two protocols that allow efficient visualization of transcripts of interests from animals with high variety in their neuronal tissue density and composition with molluscs, arthropods, and ctenophores as illustrative examples. The entire protocol takes up to 4 days. It was successfully applied both for multi...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Detection of mRNA and microRNA Expression in Basal Chordates, Amphioxus and Ascidians
In conclusion, the method described in this chapter permits the detailed visualization of gene expression at single-cell resolution and makes it a useful tool for analyzing expression patterns of neuron specific genes in both amphioxus and ascidians. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Gene Expression Analysis of Lamprey Embryos
Lampreys represent one of two extant jawless vertebrates (cyclostomes) that diverged from jawed vertebrates over 500 million years ago. They are aquatic inhabitants with elongated, eel-shaped bodies and lack paired fins. Instead of jaws, lampreys possess a disc-shaped oral funnel armored with horny teeth. Their larvae, called ammocoetes, exhibit worm-like morphology without the orbits or the oral funnel. From their unique phylogenetic position with curious morphological and developmental traits, lampreys have been widely regarded as a valuable cyclostome model, especially for the study of early vertebrate evolution. Howeve...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Analysis of Embryonic Gene Expression Patterns in the Hagfish
Because of its apparently primitive morphology and phylogenetic position, the hagfish is recognized as one of the most basal lineages of vertebrates. In particular, their embryonic gene expression patterns were expected to provide insights into the common ancestral state of the molecular developmental mechanisms. However, it has been difficult to obtain hagfish embryos because this group lives in the deep sea. To overcome this difficulty, a relatively shallow-water hagfish species (Eptatretus burgeri) has been used for embryonic gene expression analysis, and successful embryonic in situ hybridization has been reported rece...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Manual and Automated Whole-Mount In Situ Hybridization for Systematic Gene Expression Analysis in Embryonic Zebrafish Forebrain
Large-scale data collections of gene expression patterns have been assembled to construct molecular atlases of embryonic and adult vertebrate brains. These open data collections are proving useful as profound resources for developmental, physiological, and functional studies and provide a basis for deciphering the molecular anatomies of vertebrate brains. The interrogation of gene expression data from such resources allows identifying genes with restricted regional patterns and neuronal profiles. The corresponding cDNA/EST clones can be ordered from publicly accessible clone collections strongly facilitating the systematic...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
High-Throughput In Situ Hybridization: Systematical Production of Gene Expression Data and Beyond
A plethora of modern-day techniques allows the detailed characterization of the transcriptome on a quantitative level. Analyses, based on techniques such as cDNA microarrays or RNA-seq (whole transcriptome shotgun sequencing), are usually genome wide in scope and readily detect small changes in gene expression levels across different biological samples. However, when it comes to spatial localization of gene expression within the context of complex tissues, traditional methods of in situ hybridization remain unparalleled with regard to their cellular resolution. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Large-Scale ISH on Mouse Brain Sections for Systematic Gene Expression Analysis in Developing Mouse Diencephalon
The mammalian diencephalon gives rise to the thalamus and hypothalamus. The thalamus contains multiple sensory nuclei and relays sensory information to corresponding cortical areas, while the hypothalamus is a central regulator of many homeostatic behaviors that are essential for survival, such as temperature regulation, food intake, and circadian rhythms. However, despite the functional importance of these brain regions, the molecular mechanisms that guide specification and differentiation of neurons in specific thalamic and hypothalamic regions are still largely unknown. It is therefore crucial to obtain molecular marker...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
