Metastasis Research Protocols
Susan A. Brooks, Udo Schumacher
Humana Press, Jul 3, 2001 - Medical - 344 pages
Metastasis Research Protocols describes in detail all the methods needed to investigate why and how metastasis occurs. Volume I presents a comprehensive collection of established and leading-edge techniques for analyzing the expression of key molecules and for examining their production at the genetic level. The work focuses on the analysis and mapping of molecules produced by cells and tissues, and on the molecular biology underlying their expression. The traditional methods range from the histopathological and the immunocytochemical to SDS-PAGE, Western blotting, and enzyme zymography. Newer and more specialized techniques for analyzing the genetic aspects of metastasis include in situ hybridization to localize mRNAs, FISH, CGH, methylation analysis of CpG islands, RT-PCR, and differential display. Volume II presents a comprehensive collection of established and leading-edge techniques for examining metastatic behavior in vitro and whole animal models. Methods are provided for the separation of cell lines with high and low metastatic potential, along with assay systems to test defined aspects of the metastatic cascade. These systems include cell migration assays, assays for matrix degrading enzymes, basement membrane degrading assays, adhesion assays, and assays of angiogenesis. The detailed descriptions of animal models of metastasis cover the use of immunodeficient animals, syngenic and transgenic models, and orthotopic models of metastasis.
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Histopathological Assessment of Metastasis
In Vitro Assays of Metastatic Behavior
Adhesion of Tumor Cells to Matrices and Endothelium
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acid adhesion agarose aliquots analysis angiogenesis antigen approx assay avidin binding Biol blocking blotting breast cancer Cancer Res carcinoma cDNA centrifuge chromosomes concentration coverslips CpG sites culture denaturation detection dH2O dilution Discard distilled water EDTA endothelial enzyme epithelial Eppendorf tube ethanol expression filter fluorescence fluorochromes formamide fresh gelatinases gene genomic hematoxylin humid chamber hybridization Incubate integrin labeling lectin lectin buffer marker membrane metaphase metastasis Metastasis Research metastatic method methylation mg/mL microscope microvessels molecular monoclonal antibody mRNA NaCl nonspecific Note overnight paraffin wax-embedded pellet peroxidase pipet polyclonal polymerase preparation primary antibody primer probe protein Protocols reaction reagents resuspend riboprobe RNase room temperature sample secondary antibody sections sequences serum slides sodium solution Southern blotting specific staining step stored at 4°C Subheading supernatant technique transfer Tris-HCl trypsin tumor tumor cells ug/mL vascular VEGF washing buffer xylene