细胞技术

This chapter deals with tissue preparation for subsequent detection of molecules in biological samples using immunocytochemistry and transmission electron microscopy. The aim of these methods is to localize specific molecules at high resolution in order to identify their subc ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...