Main description:

This second edition volume provides detailed protocols that address the challenges of signal-transduction IHC. This book delves into chapters that discuss the nature of signal transduction phenomena and approaches to making phosphor-specific antibodies, as well as numerous bona fide methods methods on digital imaging techniques, preservation of tissue targets, multicolor detection, flow cytometry, lipophagy analysis, apoptosis, and the combination of IHC with in situ hybridization. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and comprehensive, Signal Transduction Immunohistochemistry: Methods and Protocols, Second Edition is a valuable resource to both novices and experts in other fields of biomedical research who need advice on IHC protocols to study signal transduction. This book will also be useful for researchers in academia, government labs, and the biotech industry.

Contents:

Preface...
Table of Contents...
Contributing Authors...

1. Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications
Kathy Brumbaugh, Wen-Chie Liao, J. P. Houchins, Jeff Cooper, and Steve Stoesz

2. Antibodies That Work Again and Again and Again
Kameron Simpson and Michael Browning

3. Validating Antibody Specificities for Immunohistochemistry by Protein Blotting Methods
Biji T. Kurien and R. Hal Scofield

4. Acquisition and Post-Processing of Immunohistochemical Images
Jerry Sedgewick

5. Using Digital Quantification of Stained Tissue Microarrays as a Medium-Throughput, Quantitative Method for Measuring the Kinetics of Signal Transduction
Jessica Kalra and Jennifer Baker

6. EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis
Christianne Bandeira-Melo, Ligia Almeida Paiva, Natalia R. T. Amorim, Peter F. Weller, and Patricia T. Bozza

7. Preservation of Specific Protein Signaling States using Heat Based Stabilizor System
Mats Boren

8. Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX using Primary Antibodies Raised in the Same Host Species
Jodi Hagen, David Schwartz, and Alexander E. Kalyuzhny

9. Culture, Transfection and Immunocytochemical Analysis of Primary Macrophages
Charles A. Warwick and Yuriy M. Usachev

10. Phenotyping CD4+ hTh2 Cells by Flow Cytometry: Simultaneous Detection of Transcription Factors, Secreted Cytokines and Surface Markers
Chris Goetz, Li-Jen Peng, Birte Aggeler, and Jody Bonnevier

11. A Coupled Approach Utilizing Immunohistochemistry and Immunocytochemistry to Visualize Cellular Lipophagy
Aishwarya Sathyanarayan

12. Apoptotic Bodies: Selective Detection in Extracellular Vesicles
Paul Hauser, Sha Wang, and Vladimir V. Didenko

13. Activation and Differentiation of Mesenchymal Stem Cells
Pravin J. Mishra and Debabrata Banerjee

14. High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues using Secondary Antibody Conjugated to Polymer-HRP
Michael Grahek, Ana Ptak, and Alexander E. Kalyuzhny

15. Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry
David Schwartz, Michael Grahek, Yingwei He, Wei Wang, Jennifer Nguyen, and Alexander E. Kalyuzhny

16. Dual Detection of Nucleolytic and Proteolytic Markers of Lysosomal Cell Death: DNase II-Type Breaks and Cathepsin D
Candace C. Minchew and Vladimir V. Didenko

17. Multiplex Immunohistochemistry for Mapping the Tumor Microenvironment
Jessica Kalra and Jennifer Baker

18. Validation of Anti-Mouse PDL-1 Goat Polyclonal Antibody Staining with Mouse PDL-1 In Situ Hybridization on Adjacent Sections of Cell Pellets and Mouse Tumors
Jacob Ricca, Mesruh Turkekul, Afsar Barlas, Dmitry Yarilin, Sho Fujisawa, Ning Fan, Matthew Brendel, Dmitriy Zamarin, Jedd D. Wolchok, and Katia Manova-Todorova

19. Automated Double In Situ Detection of Mouse LGR5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt
Mesruh Turkekul, Afsar Barlas, Dmitry Yarilin, Sho Fujisawa, Ning Fan, Matthew Brendel, and Katia Manova-Todorova

20. Automated High Content Screening of g--H2AX Expression in HeLa Cells
Nate Hopp, Jodi Hagen, Birte Aggeler, and Alexander E. Kalyuzhny