In Situ Hybridization Protocols (Methods in Molecular Biology, 2148)

Preface
Contents
Contributors
Part I: General Applications
Chapter 1: Guidelines for the Optimization and Validation of In Situ Hybridization
	1 Introduction
	2 Steps to Validation
		2.1 Material Preparation
		2.2 Understand Target
		2.3 Methodology
			2.3.1 Probe Generation
			2.3.2 Riboprobes
			2.3.3 Oligonucleotide Probes
			2.3.4 Pretreatment
			2.3.5 Hybridization
			2.3.6 Stringency
			2.3.7 Detection
		2.4 Troubleshooting
		2.5 Determining Specificity
		2.6 Controls
			2.6.1 Positive Controls
			2.6.2 Negative Controls
	References
Chapter 2: Overcoming Autofluorescence (AF) and Tissue Variation in Image Analysis of In Situ Hybridization
	1 Introduction
	2 Materials
	3 Methods
		3.1 Imaging of the Slides
		3.2 Analyzing Images: TMA Segmentation
		3.3 Analyzing Images: Creating the Classifier
		3.4 Analyzing Images: Creating the Analysis Algorithm
	4 Notes
	References
Part II: Methods for DNA ISH
Chapter 3: Practical Application of Fluorescent In Situ Hybridization Techniques in Clinical Diagnostic Laboratories
	1 Introduction
	2 Materials
		2.1 Buffers and Fixatives
		2.2 Ready-to-Use Commercial Reagents
		2.3 Specialist Reagents, DNA Probes
		2.4 Equipment
			2.4.1 General Laboratory Equipment
			2.4.2 Specialist Equipment
		2.5 Biological Samples to Test
	3 Methods
		3.1 Preliminary Steps
		3.2 Fixation and Pretreatment of Blood, Marrow, and Tissue Imprint Preparations
		3.3 MAA Fixed Cell Suspension Preparations
		3.4 FFPE 3 μm Section Preparations
		3.5 Pretreatment of FFPE 3 μm Sections Using Pressure Cooker and Enzyme Digestion
		3.6 Probe Preparation and Application
		3.7 Post-hybridization Stringency Wash
		3.8 Counterstain and Coverslipping
		3.9 Turning on Microscope, Lamp Power Unit, and FISH Workstation: General Points
		3.10 Examination of FISH Tests and Image Capture
		3.11 Analysis of FISH Test Results
			3.11.1 Numerical Analysis
			3.11.2 Break-Apart Analysis
			3.11.3 Dual-Fusion, Dual-Color Translocation Analysis
			3.11.4 Tricolor Rearrangement Analysis
		3.12 Signal Enumeration
		3.13 Quality Assurance
		3.14 External Quality Control
	4 Notes
	References
	Other Useful Resources
	National Guidelines
	International/European Standards
Chapter 4: Fluorescent In Situ Hybridization Using Oligonucleotide-Based Probes
	1 Introduction
	2 Materials
		2.1 MYtags Immortal Oligonucleotide Library
		2.2 MYcroarray Debubbling PCR
		2.3 In Vitro Transcription
		2.4 Reverse Transcription
		2.5 Removal of RNA
		2.6 Chromosome Spread Preparation
		2.7 Fluorescent In Situ Hybridization (FISH)
	3 Methods
		3.1 MYcroarray Debubbling PCR: First Step of Amplification
			3.1.1 PCR Purification
		3.2 In Vitro Transcription: Second Step of Amplification
			3.2.1 RNA Purification
		3.3 Reverse Transcription: Labeling Step
			3.3.1 RNA:DNA Hybrid Purification
		3.4 Removal of RNA
			3.4.1 Single-Strand DNA Probe Purification
		3.5 Chromosome Spread Preparation
		3.6 FISH
			3.6.1 Slide Pretreatment
			3.6.2 Chromosome Denaturation
			3.6.3 Probe Hybridization
			3.6.4 Stringency Washes
			3.6.5 Optional Higher Stringency Washes
			3.6.6 Probe Detection
			3.6.7 Slide Cleaning for Re-probing
	4 Notes
	References
Chapter 5: Visualizing Genome Reorganization Using 3D DNA FISH
	1 Introduction
	2 Materials
		2.1 Fosmid Probe Preparation
		2.2 Slide Preparation: Cells
		2.3 Slide Preparation: Paraffin-Embedded Tissue Sections
		2.4 FISH
	3 Methods
		3.1 Selection of Fosmid Probes Using Ensembl Browser
		3.2 Preparation of Fosmid Probes Using Alkaline Lysis Miniprep (See Note 1)
		3.3 Nick Translation
		3.4 Preparation of Slides: Cells
		3.5 Preparation of Slides: Paraffin-Embedded Tissue Sections
		3.6 3D DNA FISH
			3.6.1 Preparation
			3.6.2 Probe Preparation
			3.6.3 Slide Preparation
			3.6.4 Slide Washing
		3.7 Imaging
	4 Notes
	References
Part III: Methods for Cultured Cells
Chapter 6: MicroRNA In Situ Hybridization in Paraffin-Embedded Cultured Cells
	1 Introduction
	2 Materials
		2.1 Reagents and Buffers
		2.2 Equipment
	3 Methods
		3.1 Cell Preparation and Embedding of Cells
		3.2 Tissue Sections
		3.3 In Situ Hybridization
	4 Notes
	References
Chapter 7: Multiplexed Detection and Analysis of Low-Abundance Long Noncoding RNA Using RNAscope in Cultured Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 RNAscope
		2.3 High-Content Imaging and Analysis
	3 Methods
		3.1 Cell Culture
		3.2 RNAscope Assay
		3.3 High-Content Imaging and Analysis
	4 Notes
	References
Chapter 8: Multiplexed Quantitative In Situ Hybridization for Mammalian or Bacterial Cells in Suspension: qHCR Flow Cytometry ...
	1 Introduction
	2 Materials
		2.1 Sample Preparation
			2.1.1 Fixation and Permeabilization (All Sample Types)
			2.1.2 Bacterial Samples
			2.1.3 Mammalian Samples
		2.2 HCR Reagents
		2.3 Additional Equipment
	3 Methods
		3.1 Sample Preparation
			3.1.1 Bacterial Cells
			3.1.2 Mammalian Cells
		3.2 Detection Stage
		3.3 Amplification Stage
		3.4 Flow Cytometry
		3.5 Data Analysis
			3.5.1 Raw Single-Cell Intensities
			3.5.2 Measurement of Signal, Background, and Signal-to-Background for Transgenic Targets
			3.5.3 Measurement of Signal, Background, and Signal-to-Background for Endogenous Targets
			3.5.4 Normalized Single-Cell Intensities for Analog mRNA Relative Quantitation with qHCR Flow Cytometry
	4 Notes
	References
Chapter 9: Multiplexed Quantitative In Situ Hybridization for Mammalian Cells on a Slide: qHCR and dHCR Imaging (v3.0)
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 HCR Reagents
		2.3 Additional Reagents and Equipment
	3 Methods
		3.1 Sample Preparation
		3.2 Detection Stage
		3.3 Amplification Stage
		3.4 Confocal Microscopy
		3.5 Image Analysis
			3.5.1 Raw Pixel Intensities
			3.5.2 Measurement of Signal, Background, and Signal-to-Background for Transgenic Targets
			3.5.3 Measurement of Signal, Background, and Signal-to-Background for Endogenous Targets
			3.5.4 Normalized Voxel Intensities for qHCR Imaging: Analog mRNA Relative Quantitation with Subcellular or Single-Cell Resolut...
			3.5.5 Dot Detection and Colocalization for dHCR Imaging: Digital mRNA Absolute Quantitation with Single-molecule Resolution
	4 Notes
	References
Part IV: Methods for Wholemounts and Plant Material
Chapter 10: Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount V...
	1 Introduction
	2 Materials
		2.1 Sample Preparation
			2.1.1 Fixation and Permeabilization (All Sample Types)
			2.1.2 Whole-Mount Chicken Embryo Preparation
			2.1.3 Whole-Mount Zebrafish Embryo/Larva Preparation
			2.1.4 Whole-Mount Mouse Embryo Preparation
		2.2 HCR Reagents
		2.3 Imaging
		2.4 Equipment
	3 Methods
		3.1 Sample Preparation
			3.1.1 Whole-Mount Chicken Embryos
			3.1.2 Whole-Mount Zebrafish Embryos/Larvae
			3.1.3 Whole-Mount Mouse Embryos
		3.2 In Situ HCR Protocol
			3.2.1 Detection Stage
			3.2.2 Amplification Stage
		3.3 Sample Mounting for Microscopy
			3.3.1 Whole-Mount Chicken Embryos
			3.3.2 Whole-Mount Zebrafish Embryos/Larvae
			3.3.3 Whole-Mount Mouse Embryos
		3.4 Confocal Microscopy
		3.5 Image Analysis
			3.5.1 Raw Pixel Intensities
			3.5.2 Measurement of Signal, Background, and Signal-to-Background
			3.5.3 Measurement of Background Components
			3.5.4 Normalized Voxel Intensities for qHCR Imaging: Analog mRNA Relative Quantitation with Subcellular Resolution in an Anato...
			3.5.5 Dot Detection and Colocalization for dHCR Imaging: Digital mRNA Absolute Quantitation with Single-Molecule Resolution in...
			3.5.6 Read-out/Read-In Analysis Framework for Quantitative RNA Discovery in an Anatomical Context
	4 Notes
	References
Chapter 11: Hybridization Chain Reaction for Quantitative and Multiplex Imaging of Gene Expression in Amphioxus Embryos and Ad...
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Buffers and Solutions
	3 Methods
		3.1 Embryo Collection and Fixation
		3.2 Adult Tissue Fixation, Embedding, and Sectioning
		3.3 Amphioxus HCR In Situ Hybridization (ISH) Steps
		3.4 Whole-Mount Imaging
	4 Notes
	References
Chapter 12: RNAscope Multiplex Detection in Zebrafish
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Buffers and Solutions
		2.3 Equipment
	3 Methods
		3.1 Probe Design
		3.2 Preparation of Embryos
		3.3 Preparation of Wash Buffers
		3.4 Day 1 (1 h): Probe Hybridization
		3.5 Day 2 (7 h): RNA Detection and Signal Amplification
		3.6 Embryo Staging for Imaging
	4 Notes
	References
Chapter 13: Duplex In Situ Hybridization of Virus Nucleic Acids in Plant Tissues Using RNAscope
	1 Introduction
	2 Materials
		2.1 RNAscope Assay Reagents and Samples
		2.2 Other Reagents and Materials
		2.3 Equipment
	3 Methods
		3.1 FFPE Section Preparation
		3.2 Pretreatment
		3.3 Duplex RNAscope Assay
		3.4 Visualization
	4 Notes
	References
Part V: Automated Methods for RNA ISH
Chapter 14: Automated ISH for Validated Histological Mapping of Lowly Expressed Genes
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
			2.2.1 FFPE Tissue Preparation
			2.2.2 Automated RNAscope on Ventana Discovery Ultra (See Note 1)
		2.3 Software
	3 Methods
		3.1 FFPE Tissue Preparation Using Immersion Fixation (See Note 2)
		3.2 Probes
		3.3 Automated ISH (See Note 1)
		3.4 Quantitative Image Analysis
	4 Notes
	References
Chapter 15: Automation of Multiplexed RNAscope Single-Molecule Fluorescent In Situ Hybridization and Immunohistochemistry for ...
	1 Introduction
	2 Materials
		2.1 Manual Pre-staining Treatment of Fixed Frozen Sections
		2.2 In Situ Hybridization and Immunohistochemical Staining Using the Leica BOND RX
		2.3 Mounting and Imaging
	3 Methods
		3.1 Leica BOND RX Setup
		3.2 Experimental Design
		3.3 Manual Pre-staining Sample Preparation
		3.4 In Situ Hybridization and Immunohistochemical Staining Using the Leica BOND RX
		3.5 Leica BOND RX Run Checklist
		3.6 Mounting and Imaging
	4 Notes
	References
Chapter 16: Automated Co-in Situ Hybridization and Immunofluorescence Using Archival Tumor Tissue
	1 Introduction
	2 Materials
		2.1 Advanced Cell Diagnostics
		2.2 Roche Tissue Diagnostics
		2.3 Target IHC Antibodies
		2.4 Akoya Biosciences Inc.
		2.5 Multiplex Assay Validation Slides
		2.6 Mounting
	3 Methods
		3.1 Multiplex Assay Construction and Validation
		3.2 Validated Protocol
	4 Notes
	References
Chapter 17: Automated Five-Color Multiplex Co-detection of MicroRNA and Protein Expression in Fixed Tissue Specimens
	1 Introduction
	2 Materials
		2.1 Consumables and Bulk Leica Reagents
		2.2 General Solution and Reagents
		2.3 Buffers and Reagent Solutions (See Also Table 1 for Probes, Table 2 for Antibodies, Table 3 for Dyes)
		2.4 Tissue Processing and Slide Preparation
		2.5 Equipment for Microscopy
	3 Methods
		3.1 Programming 4-Plex ISH/IHC Assay
		3.2 Pre-hybridization Steps
		3.3 ISH Hybridization (See Table 1 for Probes)
		3.4 Sequential Detection of ISH Probes (See Table 2 for Antibodies and Table 3 for Dyes)
		3.5 Sequential Detection of Protein Markers (See Table 2 for Antibodies and Table 3 for Dyes)
		3.6 Slide Mounting (Manually)
		3.7 Image Acquisition and Computer-Assisted Image Analysis
	4 Notes
	References
Chapter 18: Mixed Multiplex Staining: Automated RNAscope and OPAL for Multiple Targets
	1 Introduction
	2 Materials
		2.1 Consumables and Ancillary Leica Reagents
		2.2 RNAscope Reagents
		2.3 Antibodies, Blocking Solutions, and Tyramides
		2.4 Universal Reagents
		2.5 General Solutions and Reagents
	3 Methods
	4 Notes
	References
Part VI: Multiplexing and Combined Methods
Chapter 19: Simultaneous Visualization of RNA and Protein Expression in Tissue Using a Combined RNAscope In Situ Hybridization...
	1 Introduction
	2 Materials
		2.1 RNAscope Assay
		2.2 Opal/TSA Dyes
		2.3 Samples
		2.4 Reagents
		2.5 Solutions
		2.6 Miscellaneous
		2.7 Equipment
	3 Methods
		3.1 FFPE Section Preparation and Deparaffinization
		3.2 Pretreatment of FFPE Sections
		3.3 RNAscope ISH Multiplex Fluorescent Assay v2 (See Note 3)
		3.4 Immunofluorescence
		3.5 Results and Interpretation
	4 Notes
	References
Chapter 20: In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Tissu...
	1 Introduction
	2 Materials
		2.1 Padlock Probe Design
		2.2 Detection Probe Design
		2.3 Oligonucleotides
		2.4 Reagents
		2.5 Buffers and Solutions
		2.6 Equipment
	3 Methods
		3.1 Tissue Preparation
		3.2 Tissue Fixation
		3.3 Reverse Transcription/cDNA Synthesis
		3.4 Padlock Probe Hybridization and Ligation
		3.5 Rolling Circle Amplification
		3.6 RCA Product Detection and Barcode Sequencing
		3.7 Image Acquisition
		3.8 Anchor Probe and Barcode-Detection Probe Stripping
		3.9 Optional: RCA Product Detection and ``5th´´ Base Sequencing
		3.10 Analysis and Results
	4 Notes
	References
Chapter 21: GeoMx RNA Assay: High Multiplex, Digital, Spatial Analysis of RNA in FFPE Tissue
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Materials for Slide Preparation
		2.3 Materials for nCounter Readout
	3 Methods
		3.1 Preparing Reagents and Equipment
		3.2 Preparing Tissues
		3.3 Slide Preparation
		3.4 Overnight In Situ Hybridization
		3.5 Perform Stringent Washes
		3.6 Apply Morphology Markers
		3.7 Using the GeoMx DSP
		3.8 GeoMx Hyb Code Setup
	4 Notes
	References
Part VII: Target Selective Methods and Single Molecule Detection
Chapter 22: In Situ Point Mutation Detection in FFPE Colorectal Cancers Using the BaseScope Assay
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
	3 Methods
		3.1 Preparation of FFPE Cell Pellets
		3.2 Preparation of FFPE Tumor Samples
		3.3 Running the BaseScope Assay
		3.4 Analysis
	4 Notes
	References
Chapter 23: Using In Situ Padlock Probe Technology to Detect mRNA Splice Variants in Tumor Cells
	1 Introduction
	2 Materials
		2.1 RT Primers, PLPs, and Detection Probes
		2.2 Cell Lines and Cell Culture Media
		2.3 Reagents, Buffers, and Solutions
		2.4 Consumables and Equipment
	3 Methods
		3.1 Seeding PC Cell Lines
		3.2 Sample Preparation
		3.3 Reverse Transcription
		3.4 Hybridization and Ligation of PLPs
		3.5 Rolling Circle Amplification
			3.5.1 Hybridization of Detection Probes
			3.5.2 Imaging
			3.5.3 Image Analysis and Quantification
	4 Notes
	References
Chapter 24: Automated One-Double-Z Pair BaseScope for CircRNA In Situ Hybridization
	1 Introduction
	2 Materials
		2.1 Tissue Samples
		2.2 Reagents
		2.3 Equipment
	3 Methods
		3.1 Preparation of Tissue Sections
		3.2 Preparation of the Ventana DISCOVERY ULTRA Instrument
		3.3 Programming the Ventana DISCOVERY ULTRA Instrument
		3.4 Running BaseScope
	4 Notes
	References
Correction to: In Situ Sequencing: A High-Throughput, Multi-Targeted Gene Expression Profiling Technique for Cell Typing in Ti
Index