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.Whole-Mount In Situ Hybridization and Immunohistochemistry in Xenopus Embryos
Xenopus is a favorable experimental model in developmental biology. With its fast and external development, high number of progeny and large size, early embryos are well suited for micromanipulation to study the function of genes with relevance to human diseases. In this chapter, we present a combined method for lineage tracing and whole-mount in situ hybridization. In addition, we present protocols for immunohistochemistry and assays to monitor the cell proliferation and apoptosis in whole embryos. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
One FISH, dFISH, Three FISH: Sensitive Methods of Whole-Mount Fluorescent In Situ Hybridization in Freshwater Planarians
As freshwater planarians (flatworms) are further developed as a model system, the most valuable tool continues to be in situ hybridization (ISH) analysis of gene expression in whole mount adult animals (WISH). Multiple hurdles have been overcome during the optimization of a standard protocol for colorimetric, single color detections. However, gene function studies on planarians have evolved to the point where virtually all investigations require the analysis of multiple RNAs, simultaneously. Thus, considerable effort by the entire planarian community has been put forth to create adequate methodologies towards this goal. He...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Combined Radioactive and Nonradioactive Double In Situ Hybridization (DISH)
This chapter describes in detail a reliable method for dual in situ hybridization (ISH) that has been validated in numerous studies identifying co-localization of high and low abundant target mRNAs in brain tissues. ISH is widely used to study the spatial distribution of a target mRNA with high anatomical resolution to localize the gene transcript in small areas and even in single cells. Both radioactive- and nonradioactive-labeled cRNA probes can be used for the detection of the complementary mRNA with high specificity and sensitivity. In order to localize two gene transcripts in the same tissue and establish co-expressio...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Exploring Brain Genoarchitecture by Single and Double Chromogenic In Situ Hybridization (ISH) and Immunohistochemistry (IHC) on Cryostat, Paraffin, or Floating Sections
This chapter is devoted to explain how to handle with brain specimens to get cryostat, paraffin, or floating vibratome sections and the way they are processed during ISH and IHC. The procedure to be selected is related to the study aim, general and rapid mapping, or detailed and high resolution analysis. We likewise place emphasis on how to block (embed) and prepare the tissue blocks for sectioning, insuring that optimal section planes are obtained. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Exploring Brain Genoarchitecture by Single and Double Chromogenic In Situ Hybridization (ISH) and Immunohistochemistry (IHC) in Whole-Mount Embryos
This chapter and 5 detail various in situ hybridization protocols perfected over time in two laboratories with widespread experience in studying brain development and comparative neuroanatomy in amphibian, avian, and mammalian animal models. The aims of our research have frequently revolved on studying high-quality maps of gene expression—genoarchitecture—in the developing or adult brain. Apart of the protocols themselves, in this chapter, detailed advice is given as well about ways to treat the brain specimens to insure little damage and good histological quality during whole-mount ISH and IHC. (Source: Spring...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Multicolored Visualization of Transcript Distributions in Drosophila Embryos
We describe here a versatile protocol for simultaneous examination of three unique mRNA expression patterns in Drosophila melanogaster embryos. Three differently labeled antisense RNA probes are hybridized together to the embryos and detected by sequential alkaline phosphatase-based immunohistochemistry. Transcript distributions are revealed by colorimetric enzymatic reactions that permit to highlight each mRNA expression pattern by a differing and contrasting cellular color precipitate. We provide tips and tricks for each critical step helpful for successful application of the tricolor whole-mount in situ hybridization (W...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
In Situ Hybridization Methods for RNA Visualization in C. elegans
A protocol is described for the detection of endogenous mRNA in whole-mount tissue using antisense RNA probes of at least 400 bases in length. The protocol takes a few days and is inexpensive, yet sensitive enough to detect low-abundance transcripts. Although optimized for the nematode C. elegans, the protocol should work on similar tissue. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
ECHO-FISH for Gene Transcript Detection in Neuronal and Other Cells and Subcellular Compartments
Fluorescence in situ hybridization (FISH) has been widely used in a variety of applications such as karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. With detection sensitivity and stringency, FISH provides detailed information on tissue-specific, cell-specific, and subcellular gene expression. Despite the versatile applications developed in both academia and clinical sectors, FISH remains a labor-intensive and time-consuming technique. Herein we describe a quick and simple FISH protocol (ECHO-FISH: exciton-controlled hybridization-sensitive fluorescent oligonucleotide-FISH...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Transglutaminase-Mediated In Situ Hybridization (TransISH) for mRNA Detection in Mammalian Tissues
In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specific hapten–antibody interaction; however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA–(enzyme)
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conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of i...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Characterizing Cellular Identity at One Cell Resolution
Cellular processes are regulated at multiple levels in mammalian cells, including regulation at transcription, posttranscription, translation, and posttranslational levels. Most of the present techniques enable us to understand these processes either by analyzing RNAs or proteins. Very few methodologies such as combined in situ hybridization and immunocytochemistry allow us to visualize RNAs and proteins simultaneously in single cells. However, low abundance of certain transcripts (mRNAs and miRNAs) impedes the available methodologies to detect them at single-cell resolution. Here, we present a new improvised method of in ...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Optimization of In Situ Hybridization and Co-expression Analysis in the Central Nervous System
Formalin-fixed paraffin-embedded (FFPE) tissues represent a treasure trove of material on the many diverse diseases of the central nervous system. Recent advances in the areas of in situ hybridization and co-expression analysis now allow for the routine detection of DNA, mRNA, microRNA, viral RNA, and proteins in these samples. Locked nucleic acid (LNA) probes have revolutionized the field of in situ hybridization by increasing the melting temperature of the DNA–DNA or cDNA/mRNA/microRA hybrids such that the probe only needs to have 20 nucleotides to achieve a strong signal and low background, even for low copy numbe...
Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news
Gene Therapy for Epilepsies
Epilepsy is an excellent indication for gene therapy due to a significant unmet need, which is the high percentage of patients with symptoms that remain inadequately relieved by the current available treatments. This chapter provides an up-to-date review on the preclinical studies assessing the potential of gene therapy for epilepsy. We also provide here a set of procedures that can be used as a starting point to evaluate potential therapeutic candidates for epilepsy gene therapy. The techniques described here include adeno-associated viral vector production, genomic titering of the vector, stereotaxic neurosurgery for int...
Source: Springer protocols feed by Neuroscience - January 10, 2015 Category: Neuroscience Source Type: news
AAV Gene Therapy Strategies for Lysosomal Storage Disorders with Central Nervous System Involvement
Gene therapy is one of the most promising approaches for the treatment of lysosomal storage disorders (LSDs). This is especially true for the 75 % of LSDs that have central nervous system (CNS) involvement, where enzyme replacement therapy (ERT), the standard of care for LSDs, is ineffective in treating the neurological features of these diseases. Recombinant adeno-associated virus (AAV) vectors have emerged as the most efficient and promising gene transfer vehicles for the CNS and in particular for LSDs. Direct infusion of AAV vectors into interconnected structures in the brain has achieved widespread distribution of vect...
Source: Springer protocols feed by Neuroscience - January 10, 2015 Category: Neuroscience Source Type: news
Gene Therapy in Spinal Muscular Atrophy (SMA) Models Using Intracerebroventricular Injection into Neonatal Mice
Successful gene therapy for neurodegenerative disorders in clinical trials depends upon the success of the gene therapy applications in preclinical models of the disease. Preclinical animal studies often proceed to larger animal models and are subsequently incorporated into the final design of the clinical trials. Recent gene therapy advancements with preclinical animal models of spinal muscular atrophy (SMA) have made the move from the bench research to an actual treatment a more achievable reality. In this chapter, we gathered the most recent gene therapy advancements in SMA animal models and discuss the possibility of g...
Source: Springer protocols feed by Neuroscience - January 10, 2015 Category: Neuroscience Source Type: news
Gene Therapy for Chronic Pain: How to Manipulate and Unravel Pain Control Circuits from the Brain?
The increasing prevalence of chronic pain imposes to search for new therapeutic approaches. Despite the increase in basic and clinical pain research during the last decades, the available analgesic drugs remained considerably unchanged. Gene therapy emerged as an important tool in the pain field. Studies in experimental pain models consisted on blockade of nociceptive transmission at the spinal cord by peripheral delivery of viral vectors (mainly replication-defective forms of herpes simplex virus type 1, HSV-1). Based on these results, clinical trials using gene therapy were successfully implemented in cancer patients wit...
Source: Springer protocols feed by Neuroscience - January 10, 2015 Category: Neuroscience Source Type: news
