Signal Transduction Immunohistochemistry

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.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Contenu

Phosphosite-Specific Antibodies: A Brief Update on Generation and Applications.- Antibodies That Work Again and Again and Again.- Validating Antibody Specificities for Immunohistochemistry by Protein Blotting Methods.- Acquisition and Post-Processing of Immunohistochemical Images.- Using Digital Quantification of Stained Tissue Microarrays as a Medium-Throughput, Quantitative Method for Measuring the Kinetics of Signal Transduction.- EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis.- Preservation of Specific Protein Signaling States using Heat Based Stabilizor System.- Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX using Primary Antibodies Raised in the Same Host Species.- Culture, Transfection and Immunocytochemical Analysis of Primary Macrophages.- Phenotyping CD4+ hTh2 Cells by Flow Cytometry: Simultaneous Detection of Transcription Factors, Secreted Cytokinesand Surface Markers.- A Coupled Approach Utilizing Immunohistochemistry and Immunocytochemistry to Visualize Cellular Lipophagy.- Apoptotic Bodies: Selective Detection in Extracellular Vesicles.- Activation and Differentiation of Mesenchymal Stem Cells.- High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues using Secondary Antibody Conjugated to Polymer-HRP.- Using Phospho-Peptides Immobilized on Magnetic Beads for Absorption Control in Immunohistochemistry.- Dual Detection of Nucleolytic and Proteolytic Markers of Lysosomal Cell Death: DNase II-Type Breaks and Cathepsin D.- Multiplex Immunohistochemistry for Mapping the Tumor Microenvironment.- 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.- Automated Double In Situ Detection of Mouse LGR5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt.- Automated High Content Screening of g--H2AX Expression in HeLa Cells.