Guidelines for the Optimization and Validation of In Situ Hybridization -- Overcoming Autofluorescence (AF) and Tissue Variation in Image Analysis of In Situ Hybridization -- Practical Application of Fluorescent In Situ Hybridization Techniques in Clinical Diagnostic Laboratories -- Fluorescent In Situ Hybridization using Oligonucleotide-Based Probes -- Visualizing Genome Reorganization using 3D DNA FISH -- MicroRNA In Situ Hybridization in Paraffin Embedded Cultured Cells -- Multiplexed Detection and Analysis of Low Abundant Abundance Long Non-Coding RNA using RNAscopeTM in Cultured Cells -- Multiplexed Quantitative In Situ Hybridization for Mammalian or Bacterial Cells in Suspension: qHCR Flow Cytometry (v3.0) -- Multiplexed Quantitative In Situ Hybridization for Mammalian Cells on a Slide: qHCR and dHCR Imaging (v3.0) -- Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution within Whole-Mount Vertebrate Embryos: qHCR and dHCR imaging (v3.0) -- Hybridization Chain Reaction for Quantitative and Multiplex Imaging of Gene Expression in Amphioxus Embryos and Adult Tissues -- RNAscopeTM Multiplex Detection in Zebrafish -- Duplex In Situ Hybridization of Virus Nucleic Acids in Plant Tissues using RNAscopeTM -- Automated ISH for Validated Histological Mapping of Lowly Expressed Genes -- Automation of Multiplexed RNAscope Single-Molecule Fluorescent In Situ Hybridization and Immunohistochemistry for Spatial Tissue Mapping -- Automated Co-In Situ Hybridization and Immunofluorescence using Archival Tumor Tissue -- Automated 5-Color Multiplex Co-Detection of MicroRNA and Protein Expression in Fixed Tissue Specimens -- Mixed Multiplex Staining: Automated RNAscopeTM and OPALTM for Multiple Targets -- Simultaneous Visualization of RNA and Protein Expression in Tissue Using a Combined RNAscopeTM In Situ Hybridization and Immunofluorescence Protocol -- In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Tissue Sections -- GeoMx™ RNA Assay: High Multiplex, Digital, Spatial Analysis of RNA in FFPE Tissue -- In Situ Point Mutation Detection in FFPE Colorectal Cancers using the BaseScope Assay -- Using In Situ Padlock Probe Technology to Detect mRNA Splice Variants in Tumor Cells -- Automated One-Double-Z Pair BaseScope™ for CircRNA In Situ Hybridization.

This fifth edition volume expands on the previous editions with updated discussions on the many new in situ hybridization (ISH) techniques used by researchers today. New developments in probe designs, detection systems, specificity and sensitivity improvements, and multiplexing combinations are explored. Chapters in this book are organized into seven sections and cover general applications; methods for DNA ISH; methods for cultured cells; methods for wholemount and plant material; automated methods for RNA; multiplexing and combined methods; and targeted selective methods and single molecule detections. 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. Cutting-edge and thorough, In Situ Hybridization Protocols, Fifth Edition is a valuable resource for both novice and expert scientists interested in learning more about this exciting and advancing field. .