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In Situ Molecular Pathology and Co-Expression Analyses - 1st Edition - ISBN: 9780124159440, 9780124160019

In Situ Molecular Pathology and Co-Expression Analyses

1st Edition

Editor: Gerard Nuovo
Hardcover ISBN: 9780124159440
eBook ISBN: 9780124160019
Imprint: Academic Press
Published Date: 7th February 2013
Page Count: 298
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In Situ Molecular Pathology and Co-Expression Analyses explains, in easy-to-understand language, simplified ways of understanding and performing in situ hybridization and immunohistochemistry tests. The book also focuses on straightforward protocols used to simultaneously detect two or more proteins/nucleic acids within intact tissue by doing co-expression analyses.

The fields of in situ hybridization and immunohistochemistry have expanded rapidly due to the use of computer-based analysis. To get the most out of these automated platforms, researchers and diagnostic biomedical investigators must have a solid understanding of the basics of in situ-based tests, protocols, and regimens for troubleshooting.

Practicing molecular pathologists, clinical chemists, and toxicologists, as well as clinicians and researchers in training, will benefit from this book's clear presentation of protocols and theoretical framework.

Key Features

  • Includes over 200 easy-to-follow experimental protocols
  • Features chapter-ending summaries of "Key Points to Remember" to bring beginners up to speed with any seasoned veteran in the field
  • Offers two chapters written by industry leaders in the fields of in situ hybridization, immunohistochemistry, and computer software for co-expression analyses


Pathologists, clinical chemists, and toxicologists; practicing clinicians, fellows, and residents in pathology

Table of Contents

  • Acknowledgments
  • 1. Introduction
    • Suggested Readings
  • 2. The Basics of Molecular Pathology
    • Introduction
    • The Structure of DNA and RNA
    • Key Terms in Molecular Biology
    • The Structure of Proteins
    • Southern Blot, Northern Blot, and Dot Blot Hybridization
    • Suggested Readings
  • 3. The Biochemical Basis of In Situ Hybridization and Immunohistochemistry
    • Introduction
    • The Biochemical Effects of Fixing Cells in Formalin
    • The Biochemical Effects of Embedding the Tissue/Cells into Paraffin
    • The Biochemical Effects of Protease Digestion (Protease Pretreatment)
    • Ionic Charge and Hydrogen Bond Potential of Cell/Breathability of the Macromolecules inside the Formalin-Fixed, Paraffin-Embedded Tissues
    • The Practical Significance of the Highly Variable “Formalin-Induced Three-Dimensional Network” on Optimizing the Signal for in Situ Hybridization and Immunohistochemistry
    • The Effect of Aging of the Tissue/Paraffin Block on the “Formalin-Induced Three-Dimensional Network” for in Situ Hybridization and Immunohistochemistry
    • Summary
    • Suggested Readings
  • 4. The Basics of In Situ Hybridization
    • Introduction
    • Step One: Fixing the Tissue or Cells
    • Step Two: Putting the Fixed Cells or Tissue on a Glass Slide
    • Step Three: Pretreatment of the Tissue for Successful in Situ Hybridization
    • Step Four: Choosing a Probe for in Situ Hybridization
    • Step Five: Denaturation and Hybridization of the Probe
    • Step Six: The Stringent Wash and in Situ Hybridization
    • Step Seven: The Detection Step of in Situ Hybridization Signal
    • Step Eight: The Counterstain for in Situ Hybridization Signal
    • Suggested Readings
  • 5. The Basics of Immunohistochemistry
    • Introduction
    • Step One: Fixing the Tissue or Cells
    • Step Two: Putting the Fixed Cells or Tissue on a Glass Slide
    • Step Three: Pretreatment of the Tissue for Successful Immunohistochemistry
    • Step Four: Choosing a Primary Antibody for Immunohistochemistry
    • Step Five: the Hybridization Step
    • Step Six: the Detection Step of Immunohistochemistry
    • Step Seven: The Counterstain for Immunohistochemistry
    • Suggested Readings
  • 6. The Basics of Histologic Interpretations of Tissues
    • Introduction
    • Part One: The Different Cell Types in Formalin-Fixed, Paraffin-Embedded Tissues
    • Terms Commonly Used to Describe Formalin-Fixed, Paraffin-Embedded Tissues
    • Specialized Stromal Cells
    • Part Two: The Differentiation between Benign and Malignant Cells in Formalin-Fixed, Paraffin-Embedded Tissues
    • Part Three: Putting it All Together—Determining the Specific Cell Types that Contain Your Target of Interest
  • 7. The Recommended Protocol for in Situ Hybridization
    • Introduction
    • Troubleshooting
    • Interpretation of Data
    • Suggested Readings
  • 8. The Recommended Protocol for Immunohistochemistry
    • Introduction
    • Troubleshooting
    • Interpretation of Data
    • Suggested Readings
  • 9. Co-Expression Analyses
    • Introduction
    • Different Methodologies for Co-Expression Analyses
    • Use Computer-Based Co-Expression
    • Protocol for Co-expression Analysis in Formalin-Fixed, Paraffin-Embedded Tissues
  • 10. PCR In Situ Hybridization and RT In Situ PCR
    • Introduction
    • RT in Situ PCR
    • A Protocol for RT in Situ PCR: First Determine the Optimal Pretreatment Conditions
    • A Protocol for RT in Situ PCR: Using the One-Step rTth System
    • PCR in Situ Hybridization
    • The Theory behind RT in Situ PCR
  • Appendix 1. Locked Nucleic Acids—Properties and Applications
    • Introduction to Locked Nucleic Acids (LNA™)
    • Applications for Locked Nucleic Acids
    • Design of LNA™ Oligonucleotides
  • Appendix 2
    • Increasing Sensitivity and Accuracy of Quantitative Immunofluorescence in FFPE Tissue with Spectral Imaging
    • Receptor Trafficking Analysis with Multi-Color Fluorescence Labeling and Multispectral Imaging
    • Automated Image Analysis Of Tumor-Infiltrating Lymphocytes
    • Automated Quantitative Image Analysis of Diabetic Nephropathy
    • Studies of Receptor Signaling and Mutations in Archival Tissue using Tissue Microarrays and Multispectral Imaging
  • Appendix 3
  • Index


No. of pages:
© Academic Press 2013
7th February 2013
Academic Press
Hardcover ISBN:
eBook ISBN:

About the Editor

Gerard Nuovo

Dr. Gerard Nuovo received his MD from the University of Vermont College of Medicine in 1983. He did his training in Anatomic Pathology at Columbia Prebyterian College of Physicians and Surgeons in NYC. He then did a two year fellowship at Columbia Presbyterian in Molecular Virology and Gynecologic Pathology under the mentorship of Drs. Saul Silverstein, Chris Crum, and Ralph Richart. Dr. Nuovo’s career has been dedicated to bridging the areas of surgical pathology and molecular pathology. He has published over 400 peer review papers in the field and written several textbooks in both Anatomic Pathology and Molecular Pathology. This textbook fulfills a strong ambition of Dr. Nuovo; to empower the next generation of researchers and diagnosticians to include in situ molecular pathology in their work by using easy-to-follow and easy-to-understand protocols for in situ hybridization, immunohistochemistry, and co-expression analyses.

Affiliations and Expertise

Ohio State University Medical Center, Columbus, OH, USA


"If you perform this type of testing in your laboratory, you must get this book. When you think all experimental approaches for in situ molecular testing methods have failed, have faith and refer to this book for the solution.", October 25, 2013
"Nuovo presents a textbook on in-situ-based molecular pathology for readers with minimum prior knowledge of molecular pathology in general and in-situ-based molecular pathology in particular."--Reference and Research Book News, August 2013

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