This sixth edition volume expands on the previous editions with a discussion of the latest methods used to detect RNA or DNA species in situ, including both short and long RNAs, and coding and non-coding RNAs. The chapters in this book are organized into five parts: RNA detection techniques; RNA and protein co-detection; 3D and wholemount ISH; spatial transcriptomics approaches; and image analysis and quantification. Some of the topics covered in this volume are detection of viral pathogens using RNAscope(TM); detection of therapeutic antisense oligonucleotides using miRNAscope assays; RNAscope HiPlex in tissue sections and wholemounts; spatial transcriptomics platforms with accompanying data processing; and various image analysis platforms for downstream quantitative assessment. 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. Authoritative and thorough, In Situ Hybridization Protocols, Sixth Edition brings together a collection of protocols, along with practical notes and recommendations, and offers researchers a comprehensive introduction to the current leading edge of in situ hybridization.