Main 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:

Part I: Tissue Processing

1. Tissue Preparation for Histochemistry: Fixation, Embedding, and Antigen Retrieval for Light Microscopy
Tim D. Hewitson, Belinda Wigg, and Gavin J. Becker

2. An Optimized RNA Extraction Method from Archival Formalin Fixed-Paraffin Embedded Tissue
Joon-Yong Chung and Stephen M. Hewitt

3. Laser Capture Microdissection and Pressure-Catapulting for the Analysis of Gene Expression in the Renal Glomerulus
Amanda J. Edgley, Renae M. Gow, and Darren J. Kelly

Part II: Staining Techniques

4. Immunofluorescence Detection of the Cytoskeleton and Extracellular Matrix in Tissue and Cultured Cells
Josiane Smith-Clerc and Boris Hinz

5. Double Immunohistochemistry with Horseradish Peroxidase and Alkaline Phosphatase Detection Systems
Vincent Sarrazy and Alexis Desmouliere

6. Retrogradely-Transported Neuronal Tracers Combined with Immunohistochemistry Using Free Floating Brain Sections
Emilio Badoer

7. High-Pressure Freezing, Chemical Fixation, and Freeze-Substitution for Immuno-Electron Microscopy
Christian Muhlfeld

8. Lectin Histochemistry for Light and Electron Microscopy
Su Ee Wong, Catherine Winbanks, Chrishan S. Samuel, and Tim D. Hewitson

9. Duplex In Situ Hybridization in the Study of Gene Co-Regulation in the Vertebrate Brain
Raphael Pinaud and Jin K. Jeong

10. Special Stains for Extracellular Matrix
Andrea Monte-Alto-Costa and Luis Cristovao de Moraes Sobrino Porto

11. Active Staining of Mouse Embryos for Magnetic Resonance Microscopy
Alexandra Petiet and G. Allan Johnson

12. Immunohistochemical Detection of Tumor Hypoxia
Richard J. Young and Andreas Moeller

13. In Situ Localization of Apoptosis Using TUNEL
Tim D. Hewitson and Ian A.Darby

Part III: Imaging Techniques

14. Use of Confocal Microscopy for 3-Dimensional Imaging of Neurons in the Spinal Cord
Martin Stebbing, Simon Potocnik, Pinglu Ye, and Emilio Badoer

15. High Resolution Confocal Imaging in Tissue
Verena C. Wimmer and Andreas Moeller

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

17. Image Analysis and Quantitative Morphology
Carlos Alberto Mandarim-de-Lacerda, Caroline Fernandes-Santos, and Marcia Barbosa Aguila