Signal Transduction Immunohistochemistry: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Top Five Considerations When Choosing an Antibody
1 Introduction
2 Overall Study Considerations
2.1 Protein History: Is Your Target New, or Has It Been Well Characterized in Published Studies?
2.2 Unique Characteristics of Target: Multiple Isoforms
2.3 Antibody Specificity and the Importance of Validation
2.4 Reagents for Testing: Tissues and Cells
2.4.1 Reagent Type
2.4.2 Reagent Availability
2.4.3 Reagent Quantity
3 Species Considerations
3.1 The Species of the Antigen Sequence
3.2 The Species of the Animal Host
3.3 The Species of the Secondary Antibody
3.4 Multiplexing: Using Multiple Species to Aid Analysis
4 Clonality Considerations
4.1 Monoclonal Antibodies
4.1.1 Monoclonal Antibody Production
4.1.2 Advantages and Disadvantages to Monoclonal Antibodies
4.2 Polyclonal Antibodies
4.2.1 Polyclonal Antibody Production
4.2.2 Advantages and Disadvantages to Polyclonal Antibodies
4.3 Recombinant Antibodies
4.3.1 Recombinant Antibody Production
4.3.2 Advantages and Disadvantages of Recombinant Antibodies
5 Testing Considerations
5.1 Application Selection
5.1.1 Immunostaining: IHC, ICC, and IF
5.1.2 Immunoblotting: Westerns
5.1.3 Antigen Extraction: IP
5.1.4 Quantitative Analyses: ELISA and Beyond
5.2 Western Blot as an Antibody Validation Technique
6 Detection Considerations
6.1 Fluorescent, Chromogenic, and Chemiluminescent Labeling
6.1.1 Fluorescent Labeling
6.1.2 Chromogenic Labeling
6.1.3 Chemiluminescence
6.2 Indirect vs. Direct Detection
6.2.1 Indirect Detection: Secondary Antibodies
6.2.2 Direct Detection: Tagged Primary Antibodies
7 Final Word
References
Chapter 2: Validating Antibody Specificities for Immunohistochemistry by Protein Blotting
1 Introduction
1.1 Efficiency of Protein Transfer
2 Membrane Supports for Protein Transfer
2.1 Nitrocellulose Membranes
2.1.1 Mechanism of Immobilization
2.1.2 Disadvantages of Nitrocellulose Membrane
2.2 Polyvinylidene Difluoride
2.2.1 Mechanism of Binding to Membranes
2.2.2 Advantages of PVDF
2.3 Activated Paper
2.4 Nylon Membranes
2.4.1 Shortcomings of Nylon
3 Antibody Evaluation
4 Antibody Considerations
4.1 Polyclonal Antibodies Versus Monoclonal Antibodies
5 Methods to Transfer Proteins from Gel to Membrane
5.1 Simple Diffusion
5.2 Vacuum Blotting
5.3 Electroblotting
5.3.1 Wet Transfer
5.3.2 ``Semidry´´ Transfer
6 Conclusion
References
Chapter 3: Recent Advances in Chromogens for Immunohistochemistry
1 Introduction
2 Single-Color IHC
3 Double-Stain IHC
4 Multiplex IHC
5 Clinical Indications
6 Clinical Applications
7 Summary
References
Chapter 4: Overview of Imaging Techniques in Immunohistochemistry
1 Introduction
1.1 Image Integrity
1.2 Applications for the Manipulation of Images
2 Materials
3 Before Any Corrections Are Done: Acquisition of Images
3.1 Saturation
3.2 Gamma
3.3 Bit Depth (Tonal Depth)
3.4 Noise
3.5 Focus
3.6 Correcting Uneven Illumination
3.7 Color Balance (Brightfield Only)
3.8 Metadata
3.9 Saving Images
4 Post-processing Correction Methods
4.1 Typical Procedure for Processing Brightfield and Darkfield Images: Changing Mode
4.2 Color to Grayscale
4.3 Bit Depth
4.4 Brightfield: Correcting Color and Brightness
4.4.1 Color Sampler Tool and the Info Box
4.4.2 Thresholding to Find Brightest Area
4.4.3 Color Balancing and Brightness
4.4.4 More About Setting the Darkest Black Value
4.5 Brightfield: Saturated Colors
4.6 Darkfield: Brightness Correct
4.7 Darkfield: Reduce Noise
4.8 Darkfield: Change Color
4.9 Brightfield/Darkfield: Correct Uneven Illumination
4.10 Darkfield: Blend Images
4.11 Extended Depth of Focus
5 Notes
5.1 History of Corrections Made
5.2 ImageJ and GIMP
5.3 Macintosh Versus PC
5.4 Changing the Minimum and Maximum Values in Levels
References
Chapter 5: Visualizing Activated Myofibroblasts Resulting from Differentiation of Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescence
3 Methods
3.1 Cell Culture
3.2 Immunofluorescence Staining of F Actin
3.3 Immunofluorescence Staining for MTOC
3.4 Exposure of hMSCs to Keratinocyte-Conditioned Medium (KCM)
3.5 Immunofluorescence Analysis for Markers of Myofibroblast Differentiation
4 Notes
References
Chapter 6: Clinical Applications of Flow Cytometry in Cancer Immunotherapies: From Diagnosis to Treatments
1 Introduction
2 Materials
2.1 RBC Lysis Buffer
2.2 Phosphate Buffer Saline (PBS)
2.3 FACS Buffer
2.4 Permeabilization Buffer
2.5 Viability Dyes
2.6 Fluorophore-Conjugated Antibodies
2.7 Cell Staining Dyes
3 Methods
3.1 Protocol for Immunostaining Using Directly Conjugated Antibodies
3.2 Protocol for Immunostaining Using Unconjugated Antibodies (Indirect Staining)
3.3 Phenotypic Evaluation of Leukemia/Lymphoma by Flow Cytometry
3.4 Rate of Proliferation and Cell Cycle Analysis
3.5 Protocol for Cell Proliferation and Cell Cycle Analysis
3.6 Staining, Sample Acquisition, and Analysis
3.7 Dye Dilution Proliferation Assays
3.8 Immune Monitoring During Cancer Treatment
3.9 Degranulation Assay
3.10 Intracellular Staining Assay
4 Notes
5 Future Scope of Flow Cytometry
References
Chapter 7: Labeling of Phospho-Specific Antibodies with oYo-Link Epitope Tags for Multiplex Immunostaining
1 Introduction
2 Materials
2.1 Antibodies and Antibody Labeling Reagents
2.2 Photocrosslinking
2.3 SDS-Page
2.4 Cell Preparation for Immunocytochemistry
2.5 Immunocytochemistry
3 Methods
3.1 Labeling of Antibodies with oYo-Link Epitope Tags
3.2 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
3.3 Cell Preparation for Immunocytochemistry
3.4 Immunocytochemistry
4 Notes
References
Chapter 8: Multiplex Immunochromogenic Tissue Staining Employing Primary Antibodies from the Same Species
1 Introduction
2 Materials
2.1 Chromogenic Multiplex Immunohistochemistry Slide Staining Using UltraPlex
2.2 Scanning UltraPlex Stained Slides
2.3 Primary and Enzyme-Conjugated Anti-Tag Antibodies
3 Methods
3.1 UltraPlex Manual Slide Staining
3.2 UltraPlex Automated Slide Staining with On-Board HIER
4 Notes
References
Chapter 9: 3D Imaging for Cleared Tissues and Thicker Samples on Confocal and Light-Sheet Microscopes
1 Introduction
2 Materials
3 Methods
3.1 3D Imaging on a Confocal Microscope
3.2 3D Imaging on a Light-Sheet Microscope
4 Notes
References
Chapter 10: Optical Tissue Clearing Enables Three-Dimensional Morphometry in Experimental Nerve Regeneration Research
1 Introduction
2 Materials
2.1 Reagents
2.2 Tested Antibodies
2.3 Solutions
2.4 Microscopy and Image Processing Material
3 Methods
3.1 Immunostaining
3.2 Optional: Agarose Embedding for Small Samples (e.g. Dorsal Root Ganglia). This Step Must Be Performed with Minimal Light E...
3.3 Tissue Clearing (Under Fume Hood!)
3.3.1 Dehydration and Delipidation
3.3.2 Refractive Index Matching for Microscopy (See Note 1)
4 Notes
References
Chapter 11: Protocol and Software for Automated Detection of Lysosome Active ``Runs´´ and ``Flights´´ with Wavelet Transform A...
1 Introduction
1.1 Spatial and Temporal Heterogeneity of Lysosome Movements
1.2 Experimental Requirements for Quantifying Lysosome Movement Patterns
1.3 Automated Detection of Lysosome ``Runs´´ and ``Flights´´ with a Wavelet-Based Approach
2 Materials
2.1 Cell Line
2.2 Cell Culture Media
2.3 Transfection Reagent
2.4 Choice of Reagents for Lysosome Labeling
2.5 Glass Bottom Dishes
2.6 Microscope
2.7 Particle Tracking Tools
2.8 Code for Continuous Wavelet Transform (CWT) Analysis of Lysosome Trajectories
3 Methods
3.1 Cell Culture and Lysosome Labeling
3.2 Live Cell Imaging
3.3 Lysosome Tracking with Fiji/TrackMate
3.4 Data Analysis
3.4.1 Getting Started
3.4.2 Active Transport Detection
4 Notes
References
Chapter 12: Phenotyping of M1 and M2a Macrophages and Differential Expression of ACE-2 on Monocytes by Flow Cytometry: Impact ...
1 Introduction
2 Materials
2.1 PBMCs
2.2 MagCellect Human CD14+ Cell Isolation Kit (Bio-Techne Catalog # MAGH105)
2.3 Cell Culture Plates
2.4 Media
2.5 Cytokines and Growth Factors
2.6 Flow Cytometry Antibodies for Macrophage/Monocyte Surface Marker Expression
2.7 Buffers
3 Methods
3.1 Magnetic Enrichment of CD14+ Monocytes
3.2 M0/M1 and M1 Human Monocyte-Derived Macrophage Differentiation and Polarization
3.3 M0 and M2a Human Monocyte-Derived Macrophage Differentiation and Polarization
3.4 Macrophage Harvest and Flow Cytometry Staining Protocol
3.5 ACE-2 Expression by Flow Cytometry in Human Whole Blood and PBMC Monocytes
4 Notes
References
Chapter 13: In Situ Hybridization (ISH) Combined with Immunocytochemistry (ICC) Co-detection of Phosphorylated EGFR in A431 Cu...
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 ICC
2.3 ISH
3 Methods
3.1 Cell Preparation
3.2 Integrated Co-detection Workflow (ICW)
4 Notes
References
Chapter 14: In Situ Hybridization (ISH) Combined with Immunohistochemistry (IHC) for Co-detection of EGFR RNA and Phosphorylat...
1 Introduction
2 Materials
2.1 IHC
2.2 ISH
3 Methods
3.1 FFPE Multi-tissue Array (MTA) Preparation
3.2 Integrated Co-detection Workflow (ICW)
4 Notes
References
Chapter 15: Multiplex Spatial Protein Detection by Combining Immunofluorescence with Immunohistochemistry
1 Introduction
2 Materials
3 Methods
3.1 Multiplex IF
3.2 Multiplex IHC
3.3 Imaging of Multiplex Slides
3.4 Computational Alignment of Slide Images
3.4.1 Prepare Images
3.4.2 Install, Configure, and Run Script
3.4.3 Working with Images
4 Notes
References
Chapter 16: Transcription Factor-Mediated Differentiation of Motor Neurons from Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Culture and Maintenance of hPSCs
2.2 NPC Generation from hPSCs
2.3 Lentivirus Package
2.4 Isolation of Primary Glia
2.5 MN Differentiation from NPCs
2.6 Immunostaining Assay
2.7 Electrophysiology
3 Methods
3.1 Culture and Maintenance of hPSCs
3.2 NPC Generation from hPSCs
3.3 Lentivirus Package
3.4 Isolation of Primary Glia
3.5 MN Differentiation from NPCs
3.6 Immunostaining Assay
3.7 Electrophysiology
4 Notes
References
Chapter 17: Immunohistochemical Techniques for Phosphoproteins
1 Introduction
2 Materials
2.1 Buffers, Diluents, Antigen Retrieval, and Antibody Elution Solutions
2.2 Peroxidase Activity and Blocking Solutions
2.3 Antibodies, Substrates, Chromogens, and Counterstain Solutions
3 Methods
3.1 Simple Staining
3.2 Double Staining: Sequential Method (See Note 1)
4 Notes
References
Chapter 18: Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in ...
1 Introduction
2 Materials
2.1 Hippocampal Neurons
2.2 Fixatives and Blocking Buffer
2.3 Antibodies
2.4 Viruses and Expression Plasmids
3 Methods
3.1 Neuronal Culture
3.2 Fixation Methods
3.3 Permeabilization and Blocking Methods
3.4 Protein Immunolabeling and Phalloidin Staining
3.5 Fluorescence Imaging
3.6 Cell Vitrification and cryoET Imaging
4 Notes
References
Chapter 19: Cyclic Multiplex Fluorescent Immunohistochemistry Protocol to Phenotype Glial Cells in Formalin-Fixed Paraffin-Emb...
1 Introduction
2 Materials
3 Methods
3.1 First Cycle of Immunohistochemistry
3.2 Imaging
3.3 Antibody Denaturing and Fluorescence Quenching
3.4 Second and Successive Cycles of Immunohistochemistry
3.5 Image Analysis
4 Notes
References
Chapter 20: Spatially Characterizing the Immune Contexture in Mouse Tissue Using Multiplex Immunohistochemistry
1 Introduction
2 Materials
2.1 Opal mIHC Kit
2.2 Antibodies, Diluent, and Reagents
2.3 Buffer Reagents
2.4 Other Reagents
3 Methods
3.1 Preparing and Processing Mouse Tissue for mIHC Staining
3.2 Preparing Reagents for mIHC
3.3 Generating a Spectral Library and Autofluorescence Slides
3.4 Preparing TBST Wash Buffer
3.5 Preparing Antigen Retrieval Buffers
3.6 Preparing Low pH Glycine-Based Stripping Buffer
3.7 mIHC Staining of Mouse Tissue Slides
4 Notes
References
Chapter 21: Highly Multiplexed Immunofluorescence Imaging for Quantitative Spatial Analysis in Tissue Samples with ChipCytomet...
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Tissue Sample
3.1.1 Tissue Sectioning
3.1.2 Antigen Retrieval
3.1.3 Preparation of CellScape Chips
3.2 Staining and Image Acquisition
4 Notes
References
Index