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Full Description
Somuchofwhatweknowaboutthepathogenesisofhumandiseasehascomefromthe systematic and careful study of histological material. Indeed, every internal medicine discipline has its landmark papers describing the clinico-pathological correlations. However, increasingly, it is molecular and cellular biology that provides the necessary mechanistic insights. For many years, it was thought that the two skill sets were mutually exclusive, but we hope that this book shows that this is not necessarily so. Implicitinthescienceofhistologyisthepreservationandarchivingoftissue.PartIof the book concentrates on the preparation of tissue, providing an overview of fixation, embedding, and processing (Chapter 1), and in Chapters 2 and 3, the required techniques for the retrieval of RNA from histological sections. Both routine and specialist histological staining techniques are provided in Part II. These include pro- cols for immuno (Chapters 4-7), lectin (Chapter 8), and hybridization (Chapter 9) histochemistry,histologicalstaining (Chapters10and11),aswellasspecificmethods for the in situ identification of hypoxia (Chapter 12) and apoptosis (Chapter 13). Finally, Part III details advances in imaging (Chapters 14-16) and image analysis (Chapter 17). It is hoped that this volume will provide molecular biologists with the basic his- chemical techniques and histologists with the molecular techniques to realise the potential of their resource. We are indebted to the authors for their generosity in sharing these protocols.
Contents
Tissue Processing.- Tissue Preparation for Histochemistry: Fixation, Embedding, and Antigen Retrieval for Light Microscopy.- An Optimized RNA Extraction Method from Archival Formalin-Fixed Paraffin-Embedded Tissue.- Laser-Capture Microdissection and Pressure Catapulting for the Analysis of Gene Expression in the Renal Glomerulus.- Staining Techniques.- Immunofluorescence Detection of the Cytoskeleton and Extracellular Matrix in Tissue and Cultured Cells.- Double Immunohistochemistry with Horseradish Peroxidase and Alkaline Phosphatase Detection Systems.- Retrogradely Transported Neuronal Tracers Combined with Immunohistochemistry Using Free-Floating Brain Sections.- High-Pressure Freezing, Chemical Fixation and Freeze-Substitution for Immuno-electron Microscopy.- Lectin Histochemistry for Light and Electron Microscopy.- Duplex In Situ Hybridization in the Study of Gene Co-regulation in the Vertebrate Brain.- Special Stains for Extracellular Matrix.- Active Staining of Mouse Embryos for Magnetic Resonance Microscopy.- Immunohistochemical Detection of Tumour Hypoxia.- In Situ Localization of Apoptosis Using TUNEL.- Imaging Techniques.- Use of Confocal Microscopy for Three-Dimensional Imaging of Neurons in the Spinal Cord.- High-Resolution Confocal Imaging in Tissue.- Software-Based Stacking Techniques to Enhance Depth of Field and Dynamic Range in Digital Photomicrography.- Image Analysis and Quantitative Morphology.
