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丁香实验推荐阅读
Visualization of mRNA Expression in the Zebrafish Embryo

Examination of spatial and temporal gene expression pattern is a key step towards understanding gene function. Therefore, in situ hybridization of mRNA is one of the most powerful and widely used �techniques in biology. Recent advances allow the reliable and simultaneous detection of mR ...

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Visualization of mRNA Localization in Xenopus Oocytes

Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. Th ...

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Localization and Anchorage of Maternal mRNAs to Cortical Structures of Ascidian Eggs and Embryos Using High Resolution In Situ Hybridization

In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and ...

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High Resolution Fluorescent In Situ Hybridization in Drosophila

Tissue-specific gene expression is a major determinant in the elaboration of cells with distinctive phenotypes and functions, which is crucial for the development and homeostasis of multicellular organisms. Fluorescent in situ hybridization (FISH) is a powerful method for asses ...

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FISH and Immunofluorescence Staining in Chlamydomonas

Here we describe how to use fluorescence in situ hybridization and immunofluorescence staining to determine the in situ distributions of specific mRNAs and proteins in Chlamydomonas reinhardtii. This unicellular eukaryotic green alga is a major model organism in cell biological r ...

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High-Resolution Fluorescence In Situ Hybridization to Detect mRNAs in Neuronal Compartments In Vitro and In Vivo

The localization of specific mRNAs into dendrites and/or axons is an important mechanism to enrich �proteins at their sites of function and influence neuronal development, plasticity, and repair. The fluorescence in situ hybridization (FISH) methods described here have provided h ...

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Localization of mRNA in Vertebrate Axonal Compartments by In Situ Hybridization

The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demons ...

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Genetically-Encoded Fluorescent Probes for Imaging Endogenous mRNA in Living Cells

Localization of mRNAs plays pivotal roles in different cell types, including neurons and the cells in the developing stages. To visualize the dynamic movements of mRNAs in living cells, many methods have been emerged in the past decade. However, it has not been realized to visualize endogenous m ...

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Delivery of Molecular Beacons for Live-Cell Imaging and Analysis of RNA

Over the past decade, a variety of oligonucleotide-based probes have been developed that allow for direct visualization of RNA molecules in living cells. Of these, molecular beacons have garnered a particularly high degree of interest due to their simple yet exquisite unimolecular stem ...

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Tiny Molecular Beacons for in vivo mRNA Detection

The molecular beacon technology is an established approach for visualizing native mRNAs in living cells. These probes need to efficiently hybridize to accessible RNA regions in order to spatially and temporally resolve the dynamic steps of the RNA life cycle. A refined method using two comp ...

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Visualization of Induced RNA in Single Bacterial Cells

Visualization of RNA in live cells is a challenging task due to the transient character of most RNA molecules and the lack of adequate methods to label RNA noninvasively. Here, we describe a system for regulated RNA synthesis and visualization of RNA in live Escherichia coli cells based on protein co ...

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Simultaneous Real-Time Imaging of Signal Oscillations Using Multiple Fluorescence-Based Reporters

It is now well understood that G protein-coupled receptor (GPCR)-mediated cell signalling is subject to extensive spatial–temporal control, and that a meaningful understanding of this complexity requires techniques to study signalling at the molecular and sub-cellular level. T ...

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Genetically Encoded Fluorescent Reporters to Visualize Protein Kinase C Activation in Live Cells

Protein kinase C (PKC) signaling drives many important cellular processes and its dysregulation results in pathophysiologies such as cancer (Gokmen-Polar et al., Cancer Res 61:1375–1381, 2001). Because PKC is activated acutely and allosterically, it is difficult to monitor the cellu ...

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Using FRET-Based Reporters to Visualize Subcellular Dynamics of Protein Kinase A Activity

The ubiquitous Protein Kinase A (PKA) signaling pathway is responsible for the regulation of numerous processes including gene expression, metabolism, cell growth, and cell proliferation. This method details how to monitor real-time PKA activity dynamics in mammalian cells using f ...

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Investigating G Protein-Coupled Receptor Endocytosis and Trafficking by TIR-FM

G protein-coupled receptors (GPCRs) represent the largest and most versatile family of signaling receptors. Their actions are highly regulated, both under physiological conditions and in response to clinically relevant drugs. A key element in this regulation is control of the number ...

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Visualizing Receptor Endocytosis and Trafficking

G-protein-coupled 7 transmembrane domain receptors (GPCR-7TMR) represent the largest class of membrane protein drug targets. They respond to a plethora of ligands ranging from small molecules to polypeptide hormones. Upon activation, almost all GPCR-7TMRs undergo desensitiz ...

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Visualizing G Protein-Coupled Receptor Signalsomes Using Confocal Immunofluorescence Microscopy

The heptahelical G protein-coupled receptors (GPCRs) receive and transmit a wide range of extracellular stimuli and induce a wide array of cellular responses by activating signaling kinases. It has become increasingly evident that the agonist-stimulated GPCR complexed with the ad ...

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Detection and Characterization of Receptor Interactions with PDZ Domains

Many transmembrane receptors are regulated by associations with scaffold proteins containing PDZ domains, which interact with receptor carboxyl-termini to control receptor signaling, trafficking, and localization. We describe here approaches for detecting and charac ...

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Tandem Affinity Purification and Identification of Heterotrimeric G Protein-Associated Proteins

Heterotrimeric G proteins are the main signal-transducing molecules activated by G protein-coupled receptors. Their GTP-dependent activation leads to the regulation of different effectors such as adenylyl cyclases, phospholipases, and RhoGEFs. To understand the full biol ...

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Study of G Protein-Coupled Receptor/-arrestin Interactions Within Endosomes Using FRAP

β-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of β-arrestin/receptor interactions and how these complexes, and complexes with ot ...

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