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Duplex In Situ Hybridization in the Study of Gene Co-regulation in the Vertebrate Brain

We describe here a high-sensitivity in situ hybridization protocol, optimized for fresh-frozen brain sections, that enables the detection of two transcripts, at single cell resolution. Riboprobes directed against two mRNAs of interest are synthesized with nucleotides tagged wi ...

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Lectin Histochemistry for Light and Electron Microscopy

Glycoconjugates are complex macromolecules present in all tissues throughout the body. Depending on the tissue region, glycoconjugates express different carbohydrate moieties, which can be used to both distinguish cell type and examine changes in cell phenotype. Although the pe ...

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Use of Confocal Microscopy for Three-Dimensional Imaging of Neurons in the Spinal Cord

Confocal microscopy provides a powerful and efficient tool for studying the morphology of cells. Here we describe its use to study the morphology of neurons in the dorsal horn of the spinal cord either following electrophysiological studies in live tissue slices or in neurons filled with dye in ...

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Software-Based Stacking Techniques to Enhance Depth of Field and Dynamic Range in Digital Photomicrography

Several software solutions are powerful tools to enhance the depth of field and improve focus in digital photomicrography. By these means, the focal depth can be fundamentally optimized so that three-dimensional structures within specimens can be documented with superior quality. T ...

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High-Resolution Confocal Imaging in Tissue

Laser scanning microscopy is playing a major role in visualization of biological structures and processes. However, as images are degraded due to blurring, noise, and color shifts, quantitative interpretation of confocal images can be difficult. In this chapter, we detail a procedure th ...

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Image Analysis and Quantitative Morphology

Quantitative studies are increasingly found in the literature, particularly in the fields of development/evolution, pathology, and neurosciences. Image digitalization converts tissue images into a numeric form by dividing them into very small regions termed picture elemen ...

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Neoepitope Antibodies Against MMP-Cleaved and Aggrecanase-Cleaved Aggrecan

Neoepitope antibodies recognize the newly created N or C terminus of protein degradation products but fail to recognize the same sequence of amino acids present in intact or undigested protein. Aggrecan neoepitope antibodies have been pivotal in studies determining the contributi ...

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Near-Infrared Optical Proteolytic Beacons for In Vivo Imaging of Matrix Metalloproteinase Activity

The exuberant expression of proteinases by tumor cells has long been associated with the breakdown of the extracellular matrix, tumor invasion, and metastasis to distant organs. There are both epidemiological and experimental data that support a causative role for proteinases of the m ...

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Zymography and Reverse Zymography for Detecting MMPs and TIMPs

Zymography is the electrophoretic separation of proteins through a polyacrylamide gel containing a proteolytic substrate. After denaturing (but nonreducing) electrophoresis, proteins are renatured and incubated in an appropriate buffer for proteolytic activity. Clear ...

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Real-Time PCR Expression Profiling of MMPs and TIMPs

Quantitative reverse transcriptase polymerase chain reaction enables the accurate quantification of gene expression in cultured cells or small tissue samples. In this chapter, we describe the use of Taqman� technology to measure expression of matrix metalloproteinases and re ...

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Single Nucleotide Polymorphism Genotyping in MMP Genes: The 5 Nuclease Assay

Many molecular genetic studies of human diseases involve determining the genotypes for single nucleotide polymorphisms. This chapter summarises a number of different techniques for the single nucleotide polymorphism genotyping which can be applied to MMP genes. The chapter also ...

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Analysis of the Degradome with the CLIP-CHIP Microarray

The degradome microarray – CLIP-CHIP™ – is a dedicated and focused array that allows the analysis of all proteases, non-proteolytic homologs, and protease inhibitor gene transcripts in the human and murine genomes at the mRNA transcript level. Based on unique 70-mer oligonucleotides, des ...

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In Vitro Model of Cartilage Degradation

This 14-day model of cartilage breakdown involves stimulation of bovine nasal cartilage with a combination of interleukin-1 and oncostatin M. Media is harvested on days 7 and 14 and the conditioned media and remaining cartilage at day 14 assayed for the levels of proteoglycan and collagen fra ...

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Immunoassays for Collagenase-Mediated Cleavage of Type I and II Collagens

This chapter describes the production and characterization of antibodies raised against neoepitopes in collagenase-cleaved collagen. It also details the development, validation, and use of immunoassays using such antibodies to measure specifically collagenase-medi ...

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Analysis of MMP-Dependent Cell Migration and Invasion

Analysing cell migration and invasion is of interest to many investigators as they mimic a part of physiological or pathological events. In this chapter, methods to analyse MMP-dependent 2D and 3D cell migration are described in detail. To study 2D cell migration, the phagokinetic track moti ...

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Kinetic Analysis of the Inhibition of Matrix Metalloproteinases: Lessons from the Study of Tissue Inhibitors of Metalloproteinases

Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and “tight-binding” nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges ...

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Identification of Cellular MMP Substrates Using Quantitative Proteomics: Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolut

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cell ...

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Mechanism-Based Profiling of MMPs

The recognition that the successful clinical use of MMP inhibitors will require quantitative correlation of MMP activity with disease type, and to disease progression, has stimulated intensive effort toward the development of sensitive assay methods, improved analytical meth ...

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Metalloproteases and the Degradome

Metalloproteases comprise a heterogeneous group of proteolytic enzymes whose main characteristic is the utilization of a metal ion to polarize a water molecule and perform hydrolytic reactions. These enzymes represent the most densely populated catalytic class of proteases in m ...

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Expression of Recombinant MMP-28 in Mammalian Cells

The expression of a recombinant MMP in a mammalian cell line can be useful, e.g., for purification of the enzyme, to characterize function of the enzyme, or to uncover its substrates. In this chapter, we have therefore documented our experience with the recently discovered MMP-28.

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