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Why not share! Embed Size px. Start on. Show related SlideShares at end. WordPress Shortcode. ShirleenDua Follow. Published in: Science. In our method, PCR-amplified. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue s expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate.
The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis. RNA-sequencing RNA-seq measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context.
In contrast, in situ hybridization provides the location of gene expression, but only for a small number of. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope.
Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. Here we describe a method for sensitive and specific histological detection of microRNAs miRNAs by in situ hybridization. The protocol focuses on the use of locked nucleic acids LNAs , which are bi-cyclic RNA analogs that allow a significant. The protocol focuses on the use of locked nucleic acids LNAs , which are bi-cyclic RNA analogs that allow a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection.
The protocol is optimized for cryosections in order to study the spatial and temporal expression of miRNAs with high sensitivity and resolution. We detail how to construct probes, set up and conduct an LNA in situ hybridization experiment. In addition, we discuss alternative colorimetric strategies that can be used to effectively detect and visualize miRNAs including double staining with other markers. Chromatin immunoprecipitation assays have contributed greatly to our understanding of the role of histone modifications in gene regulation.
However, they do not permit analysis with single-cell resolution, thus confounding analyses of heterogeneous. However, they do not permit analysis with single-cell resolution, thus confounding analyses of heterogeneous cell populations. Here we present a method that permits visualization of histone modifications of single genomic loci with single-cell resolution in formaldehyde-fixed paraffin-embedded tissue sections based on combined use of in situ hybridization and proximity ligation assays.
We show that dimethylation of lysine 4 of histone H3 H3K4me2 at the MYH11 locus is restricted to the smooth muscle cell SMC lineage in human and mouse tissue sections and that the mark persists even in phenotypically modulated SMC in atherosclerotic lesions that show no detectable expression of SMC marker genes. This methodology has promise for broad applications in the study of epigenetic mechanisms in complex multicellular tissues in development and disease. The use of biodegradable polymeric nanoparticles NPs for controlled drug delivery has shown significant therapeutic potential.
Concurrently, targeted delivery technologies are becoming increasingly important as a scientific area of investigation. In cancer, targeted polymeric NPs can be used to deliver chemotherapies to tumor cells with greater efficacy and reduced cytotoxicity on peripheral healthy tissues. In this chapter, we describe the methods of 1 preparation and characterization of drug-encapsulated polymeric NPs formulated with biocompatible and biodegradable poly D,L-lactic- co -glycolic acid -poly ethylene glycol PLGA- b -PEG copolymers; 2 surface functionalization of the polymeric NPs with the A10 2'-fluoropyrimidine ribonucleic acid RNA aptamers that recognize the prostate-specific membrane antigen PSMA on prostate cancer cells; and 3 evaluation of the binding properties of these targeted polymeric NPs to PSMA-expressing prostate cancer cells in vitro and in vivo.
These methods may contribute to the development of other useful polymeric NPs to deliver a spectrum of chemotherapeutic, diagnostic, and imaging agents for various applications.
Conventional in situ hybridization protocols lead to loss of microRNAs, which diffuse out of the formaldehyde-fixed sample owing to their small size. Adding a carbodiimide that stably links the microRNA with the protein matrix around it prevents this. Adding a carbodiimide that stably links the microRNA with the protein matrix around it prevents this diffusion and allows detection of miRNAs at very low expression levels.
A method for preparing hard tissue sections by using an adhesive film is described. The method produces very thin to one-micrometer thick frozen sections from adult mouse and rat bone.
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The bone tissue is freeze-embedded with water-soluble medium. The bone tissue is freeze-embedded with water-soluble medium and then cut with a disposable tungsten carbide blade after mounting the adhesive film onto the cut surface. The sections are stained on the adhesive film and preserved between the adhesive film and glass slide. All the steps including the embedding, cutting, staining, and mounting are completed within only 20 min.
The soft and hard tissues are preserved satisfactorily and the bone marrow is also preserved perfectly. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly identified, and the osteoid layer of bone is clearly observed. The sections are applicable to many types of staining such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization.
The immunohistochemistry can be carried out with nonfixed and undecalcified sections.
Nucleic Acids Hybridization - Modern Applications (Electronic book text, New ed.)
In addition to these applications, the sections are used for observing the PGF fluorescence. The sections are also usable for studying the distribution of water-soluble materials in the tissues. Furthermore, the sections are very useful for gene analysis using LMD technique and for imaging mass spectrometry. Structurally, the carrageenans are a complex group of polysaccharides made up of repeating galactose-related monomers and are of three main types; lambda, kappa, and iota see Chapter 33 , Note 1.
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Each has their own gel characteristics which are all thermally reversible. The lambda form does not gel strongly at room temperature and is injectable to induce an inflammatory response. Inflammation induced by carrageenan, originally described by Winter 1 , is acute, nonimmune, well-researched, and highly reproducible.
Cardinal signs of inflammation—edema, hyperalgesia, and erythema—develop immediately following subcutaneous injection, resulting from action of proinflammatory agents—bradykinin, histamine, tachykinins, complement and reactive oxygen, and nitrogen species. Such agents can be generated in situ at the site of insult or by infiltrating cells. Neutrophils readily migrate to sites of inflammation and can generate proinflammatory reactive oxygen and other species.
The inflammatory response is usually quantified by increase in paw size edema which is maximal around 5 h postcarrageenan injection see Fig. Inflammatory response post-carrageenan injection. The ability to determine spatial and temporal microRNA miRNA accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization FISH.
Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.
This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromochloroindolyl phosphate BCIP to 5-bromochloroindole and inorganic phosphate.
This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8, expression patterns that are available at the zebrafish information network ZFIN. RNA in situ hybridization has an important place in matrix biology, as the only method that allows for in situ discrimination of precise spatial and temporal patterns of gene expression.
Whereas immunohistochemistry shows where a matrix protein.