Signal Transduction Immunohistochemistry: Methods and Protocols (Methods in Molecular Biology)

Unlike detecting constitutively expressed targets, immunohistochemical detection of labile, low abundance, and short-lived signal transduction molecules can be a very difficult task.  In Signal Transduction Immunohistochemistry: Methods and Protocols, IHC experts contribute detailed protocols addressing the numerous challenges of signal-transduction immunohistochemistry (ST-IHC).  Beginning with a set of introductory chapters, the volume moves on to cover techniques used for the preservation of antigens and their unmasking, protocols in digital imaging and image analysis of stained cells and tissues, high-throughput data collection and data analysis, and techniques used in neuroscience as well as cancer and stem cell research.  Written in the highly successful Methods in Molecular Biology series format, chapters include brief 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.  Authoritative and practical, Signal Transduction Immunohistochemistry: Methods and Protocols serves as an ideal guide for novices and as a bastion of inspiring ideas to be exploited by experienced researchers on the lookout for new experimental tricks and hints. 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 Born   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, Jillian Frisch, Michael Grahek, Chun Wu, Yingwei He, Wei Wang, Jennifer Nguyen, David Schwartz, and Alexander E. Kalyuzhny