Signal Transduction Immunohistochemistry: Methods and Protocols

Part I: Antibodies as a Tool: From Concept to Design and Application

1. Overview of the Generation, Validation, and Application of Phosphosite-Specific Antibodies

Kathy Brumbaugh, Wade Johnson, Wen-Chieh Liao, Mong-Shang Lin, J. P. Houchins, Jeff Cooper, Steven Stoesz, and Roberto Campos-Gonzalez

2. Selection and Validation of Antibodies for Signal Transduction Immunohistochemistry

Juraj Bodo and Eric D. His

3. An Overview of Western Blotting for Determining Antibody Specificities for Immunohistochemistry

Biji T. Kurien, Yaser Dorri, Skyler Dillon, Anil Dsouza, and R. Hal Scofield

4. Optimized Protocol to Make Phospho-Specific Antibodies that Work

Amy J. Archuleta, Crystal A. Stutzke, Kristin M. Nixon, and Michael D. Browning

Part II: Preservation and Unmasking of Tissue Antigens

5. Methodology and Technology for Stabilization of Specific States of Signal Transduction Proteins

Mats Bor?n

6. An Enhanced Antigen Retrieval Protocol for Immunohistochemical Staining of Formalin-Fixed, Paraffin-Embedded Tissues

Sergei I. Syrbu and Michael B. Cohen

Part III: Imaging Techniques and High-Throughput Data Analysis

7. Imaging Techniques in Signal Transduction IHC

Jerry Sedgewick

8. Practical Considerations of Image Analysis and Quantification of Signal Transduction IHC Staining

Michael Grunkin, Jakob Raundahl, and Niels T. Foged

9. Flow Cytometric Analysis of Cell Signaling Proteins

Maria A. Suni and Vernon C. Maino

10. CytoSys: A Tool for Extracting Cell Cycle-Related Expression Dynamics from Static Data

Jayant Avva, Michael C. Weis, Radina P. Soebiyanto, James W. Jacobberger, and Sree N. Sreenath

Part IV: Neuroscience, Cancer, and Stem Cell Research

11. Signaling Events Initiated by Kappa Opioid Receptor Activation: Quantification and Immunolocalization Using Phospho-Selective KOR, p38-MAPK, and KIR 3.1 Antibodies

Julia C. Lemos, Clarisse Roth, and Charles Chavkin

12. Immunohistochemical Assessment of Signal Transduction and Cell-Cycle Networks in Neural Tumors

Daniel Ciznadija, Afsar Barlas, and Katia Manova

13. Novel Multi-Color Immunofluorescence Technique Using Primary Antibodies Raised in the Same Host Species

Jillian Frisch, J.P. Houchins, Michael Grahek, Jordan Schoephoerster, Jodi Hagen, Joseph Sweet, Leopoldo Mendoza, David Schwartz, and Alexander E. Kalyuzhny

14. Activation and Differentiation of Mesenchymal Stem Cells

Pravin J. Mishra and Debabrata Banerjee

Part V: Novel Assays and Techniques

15. Double In situ Detection of Sonic Hedgehog mRNA and pMAPK Protein in Examining the Cell Proliferation Signaling Pathway in Mouse Embryo

Sho Fujisawa, Mesruh Turkekul, Afsar Barlas, Ning Fan, and Katia Manova

16. Identifying Intracellular Sites of Eicosanoid Lipid Mediator Synthesis with EicosaCell Assays

Christianne Bandeira-Melo1, Peter F. Weller2, and Patricia T. BozzaChristianne Bandeira-Melo, Peter F. Weller, and Patricia T. Bozza

17. Absorption Control in Immunohistochemistry Using Phospho-Peptides Immobilized on Magnetic Beads

Jordan Schoephoerster, Jil