Immunohistochemistry and Immunocytochemistry: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Introduction to Immunohistochemistry: From to Evolving Science to Timeless Art
	1 Introduction
	2 Antibody Structure
	3 Immunofluorescence
	4 Immunoperoxidase
	5 Indirect Immunohistochemistry
	6 Avidin-Biotin (Streptavidin) Complexes
	7 Monoclonal Antibodies
	8 Heat-Induced Antigen Retrieval
	9 Tyramide Signal Amplification
	10 Rolling Circle Amplification and In Situ Proximity Ligation
	11 Confocal Microscopy and Quantum Dots
	12 Spectral Microscopy and Multiplexing
	13 Final Remarks
	References
Chapter 2: Immunohistochemistry in Historical Perspective: Knowing the Past to Understand the Present
	1 Introduction
	2 History of Immunohistochemistry
	3 The Hybridoma Era
	4 Epilogue
	References
Chapter 3: Sample Preparation and Fixation for Histology and Pathology
	1 What Do We Expect from an Immunohistochemical Study?
	2 The Problem of the Representativity of the Sample
	3 On Sample Fixation
	4 Formaldehyde, 10% Formalin, and Neutral Buffered Formalin
		4.1 Neutral Buffered Formalin
	5 The Histological Technique Context
	6 On Sample Preparation
	7 Key Events in Sample Preparation
		7.1 Obtaining the Sample
		7.2 Primary Fixation in Neutral Buffered Formalin
		7.3 Sending the Sample to the Pathology Laboratory
		7.4 Reception and Registration in the Pathology Laboratory
		7.5 Review of the Conditions in Which the Sample Arrives and Correction of Cases
		7.6 Macroscopic Study of the Sample
		7.7 Shipment of Encapsulated Representative Sections to the Histology Laboratory
		7.8 Automatic Processing of Samples
		7.9 Paraffin Embedding
		7.10 Obtaining Paraffin Sections
		7.11 Performance of the Enzymatic Immunohistochemical Technique
	8 Conclusions
	9 Points to Remember
	References
Chapter 4: Tissue Processing
	1 Introduction
		1.1 Principles of Dehydration
		1.2 Principles of Clearing
		1.3 Principles of Infiltration
		1.4 Conventional Tissue Processing: Manual and Automated
		1.5 Microwave-Assisted Tissue Processing
	2 Materials
		2.1 Equipment
		2.2 Reagents
		2.3 Safety
	3 Methods
	4 Notes
	References
Chapter 5: Antigen Retrieval and Signal Amplification
	1 Introduction
	2 Materials
		2.1 Slide Processing
		2.2 Antigen Retrieval
		2.3 Signal Amplification
	3 Methods
		3.1 Slide Preprocessing
		3.2 Antigen Retrieval
			3.2.1 Heat-Induced Epitope Retrieval (HIER)
			3.2.2 Protease-Induced Epitope Retrieval (See Note 7)
		3.3 Amplification (See Note 8)
			3.3.1 Amplification Using Biotinylated Secondary Antibodies and ABC
			3.3.2 Tyramide Signal Amplification
	4 Notes
	References
Chapter 6: Primary Antibody Selection and Blocking Techniques for Immunohistochemistry
	1 Introduction
		1.1 Techniques
			1.1.1 Immunofluorescence
			1.1.2 Immunoperoxidase
		1.2 Primary Antibodies
			1.2.1 Polyclonal Antibodies
			1.2.2 Monoclonal Antibodies
			1.2.3 Rabbit Monoclonal Antibodies
			1.2.4 Regulatory Classifications
		1.3 Blocking Procedures
			1.3.1 Endogenous Peroxidases
			1.3.2 Endogenous Alkaline Phosphatases
			1.3.3 Endogenous Biotin
			1.3.4 Nonspecific Proteins
	2 Materials
		2.1 TRIS-Saline
		2.2 Primary Antibody Diluent
		2.3 Endogenous Peroxidase Blocking
		2.4 Alkaline Phosphatase Blocking
		2.5 Biotin Blocking
		2.6 Nonspecific Protein Blocking
	3 Methods
		3.1 Technique Selection
		3.2 Primary Antibody Selection
		3.3 Blocking Procedures
			3.3.1 Endogenous Peroxidases
			3.3.2 Endogenous Alkaline Phosphatases
			3.3.3 Endogenous Biotin
			3.3.4 Nonspecific Proteins
	4 Notes
	References
Chapter 7: Probes for Fluorescent Visualization of Specific Cellular Organelles
	1 Introduction
		1.1 Key Considerations Prior to Selecting an Organelle Dye
			1.1.1 What Specific Fluorescent Imaging Equipment Is Available?
			1.1.2 What Excitation and Emission Sources or Filters Are Available on the Microscope?
			1.1.3 What Are the Spectral Properties of the Fluorophore?
			1.1.4 Will the Sample Be Multiplexed for Probing Other Organelles or Proteins?
			1.1.5 How Does the Fluorophore Function to Label Organelle?
			1.1.6 What Is the Membrane Permeability of the Fluorophore?
			1.1.7 Is the Fluorophore Fixable?
		1.2 Organelle Specific Fluorescent Probes
			1.2.1 Nucleus
			1.2.2 Endoplasmic Reticulum
			1.2.3 Golgi Apparatus
			1.2.4 Mitochondria
			1.2.5 Lysosomes
			1.2.6 Cytoskeletal Network
	2 Materials
		2.1 Cell-Line or Cell-Type of Choice
		2.2 Appropriate Cell Culture Medium with Respective Supplements for the Chosen Cell-Line
		2.3 Dulbecco´s Phosphate Buffered Saline (Dulbecco´s PBS) pH = 7.4
		2.4 Hanks´s Balanced Salt Solution with Calcium and Magnesium (HBSS+)
		2.5 #1.5 German Glass Coverslips 0.17 mm Thickness
		2.6 Polylysine Coating Solution
		2.7 2-4% Formaldehyde Fixative, methanol Free
		2.8 0.1-1.0% Triton X-100 Cellular Permeabilization Solution
		2.9 Organelle Specific Fluorophore Probes as Noted Within each Section with Spectral Properties Matched to Available Imaging E...
		2.10 Fluorescent Imaging Equipment and Excitation/Emission Sets Compatible with Chosen Fluorophores
	3 Methods
		3.1 Attachment and Growth of Adherent Cells on Coverslips
		3.2 Formaldehyde Fixation of Cells
		3.3 Permeabilization and Blocking of Cells
		3.4 Counterstaining Nuclei with Hoechst 33342
		3.5 Staining the Endoplasmic Reticulum in Fixed Cells with Concanavalin A
		3.6 Labeling the Golgi Apparatus in Fixed Cells with Lectin GS-II
		3.7 Labeling the Mitochondria with MitoTracker
		3.8 Staining Lysosomes Using LysoTracker Probes
		3.9 Staining Plasma Membranes Using Fluorescent Wheat Germ Agglutinins
		3.10 Staining F-Actin Using Fluorescent Phalloidins
		3.11 Protecting Fluorophore Signal with Antifade Mounting Media
	4 Notes
	References
Chapter 8: Enzymatic Immunohistochemistry
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 9: Fluorescent Immunohistochemistry
	1 Introduction
		1.1 Direct and Indirect Immunofluorescence
		1.2 Principle of Fluorescence
		1.3 Photobleaching
		1.4 Autofluorescence and Background Staining
		1.5 Fluorescence Overlap
		1.6 Applications of IF in Pathology
	2 Materials
	3 Methods
		3.1 Direct Immunofluorescence
		3.2 Indirect Immunofluorescence
		3.3 Immunofluorescence of Fresh-Frozen Tissues
		3.4 Immunofluorescence of Formalin-Fixed, Paraffin-Embedded Tissues
		3.5 Controls
	4 Antigen Retrieval
		4.1 Pronase Antigen Retrieval-Immunofluorescence Procedure
		4.2 Proteinase K Antigen Retrieval-Immunofluorescence Procedure
		4.3 Trypsin Antigen Retrieval-Immunofluorescence Procedure
	5 Notes
	References
Chapter 10: Double Labeling Fluorescent Immunocytochemistry
	1 Introduction
	2 Materials
		2.1 Cells Plating and Treatment with Rapamycin and Chloroquine (See Note 1)
		2.2 Labeling
	3 Methods
		3.1 Cells Plating and Treatment with Rapamycin and Chloroquine (See Note 1)
		3.2 Labeling
	4 Notes
	References
Chapter 11: Multiplexing and Spectral Microscopy
	1 Introduction
	2 Enzymatic Multiplexed Immunohistochemistry
		2.1 Materials
			2.1.1 Slide Preprocessing and Antigen Retrieval
			2.1.2 Blocking
			2.1.3 Primary Antibodies, Secondary Antibodies, and Chromogens
			2.1.4 Counter-Staining and Mounting
		2.2 Methods
			2.2.1 Slide Preprocessing
			2.2.2 Two Primaries Raised in Different Species (Mouse-Rabbit Combination)
			2.2.3 Two Primaries Raised in the Same Species (Mouse-Mouse or Rabbit-Rabbit Combinations)
			2.2.4 Triple Labeling
			2.2.5 Quadruple Labeling
		2.3 Notes
	3 Fluorescent Multiplexed Immunohistochemistry
		3.1 Materials
			3.1.1 Slide Preprocessing
			3.1.2 Microwave Treatment
			3.1.3 Blocking and Antibodies
			3.1.4 Counterstain and Mounting
		3.2 Methods
			3.2.1 Slide Preprocessing
			3.2.2 Labeling and Signal Generation for Seven-Color Manual
		3.3 Notes
	References
Chapter 12: Fluorescent In Situ Hybridization (FISH)
	1 Introduction
		1.1 FISH Analysis
	2 Materials
		2.1 Reagent Preparation
		2.2 Cell Pellet Preparation
		2.3 FISH Hybridization and Posthybridization Wash
	3 Methods
		3.1 Reagent Preparation
		3.2 Cell Pellet Preparation
		3.3 FISH Hybridization and Posthybridization Wash
	4 Notes
	References
Chapter 13: Proximity Ligation Assay (PLA)
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Preparation
		3.2 Antibody Labeling
		3.3 Ligation and Amplification
		3.4 Image Analysis
	4 Background Information
	5 Critical Parameters
	6 Troubleshooting
		6.1 High Background Signal
			6.1.1 Blocking
			6.1.2 Primary Antibodies
			6.1.3 Washing
			6.1.4 Incubation
		6.2 Low or Lack of Positive Signal
			6.2.1 Cell Density
			6.2.2 Primary Antibodies
			6.2.3 Incubation Temperatures
			6.2.4 Use of Wash Buffers
			6.2.5 Reagent Storage and Activity
			6.2.6 Filter Used for Acquisition
		6.3 Poor Imaging or Signal Coalescence
			6.3.1 Primary Antibodies
			6.3.2 Amplification Duration
			6.3.3 Image Capture
				Understanding Results
				Time Considerations
	References
		Headings0005204107
Chapter 14: Immunohistochemistry, Quality Control, and Principles of Validation in the Central Nervous System
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preanalytic Phase
			3.1.1 Fixation and Cold Ischemia
			3.1.2 Microtomy and Slide Preparation
			3.1.3 Dewaxing and Hydrating
		3.2 Analytic Phase
			3.2.1 Epitope Retrieval
			3.2.2 Blocking Nonspecific Binding
				Blocking Method
			3.2.3 Antibodies
				Primary Antibodies
					Method
			3.2.4 Secondary Antibody/Detection System
				Method
			3.2.5 Chromogen and Substrates
				Method
			3.2.6 Counterstaining
				Method
			3.2.7 Controls
		3.3 Postanalytic Phase, Quality Control, and Principles of Validation
			3.3.1 Interpretation
			3.3.2 Quality Management
				Principles of Analytic Validation
	4 Notes
	References
Chapter 15: Culture and Phenotyping of Glial Cell Cultures, Gliospheres, and Neurospheres
	1 Introduction
	2 Materials
		2.1 Tissue Culture Equipment
		2.2 Culture of Neurospheres
		2.3 Medium Components
		2.4 Immunocytochemistry Reagents
	3 Methods
		3.1 Isolation of Neural Stem Cells
		3.2 Passaging of Neurosphere Cultures
		3.3 Differentiation of Neurospheres
			3.3.1 Coat Chamber Slides or Coverslips with Poly-l-Ornithine/Laminin
			3.3.2 Differentiation of Intact Neurospheres
			3.3.3 Differentiation of Dissociated Neurospheres
			3.3.4 Culture Conditions to Preferentially Differentiate Neural Stem Cells into Neurons
			3.3.5 Culture Conditions to Preferentially Differentiate Neural Stem Cells into Astrocytes
			3.3.6 Culture Conditions to Preferentially Differentiate Neural Stem Cells into Oligodendrocytes
	4 Immunocytochemical Analysis of Cells Derived from Differentiated Neurospheres
		4.1 Preparing Paraformaldehyde Fixing Solution
		4.2 Fixation and Permeabilization of Differentiated Cells
			4.2.1 Fixation with Paraformaldehyde
			4.2.2 Fixation with Methanol
			4.2.3 Fixation with Acetone
		4.3 Immunolabeling of Fixed Cells
	5 Immunocytochemical Analysis of Intact, Undifferentiated Neurospheres
		5.1 Fixation and Permeabilization of Intact Neurospheres
	6 Neurospheres Generated from Brain Tumor Stem Cells
		6.1 Coat Tissue Culture Dishes with Poly-HEMA
	7 Notes
	References
Chapter 16: ER+ Breast Cancer Mammosphere Formation and Analysis
	1 Introduction
	2 Materials
		2.1 Primary Mammosphere Formation: Estrogen Containing or Estrogen-Free
		2.2 Secondary Mammosphere Formation: Estrogen Containing or Estrogen-Free
		2.3 Mammosphere Formation in Matrices: Estrogen-Containing or Estrogen-Free
		2.4 Imaging and Counting of Mammospheres: Manual Method
		2.5 Imaging and Counting of Mammospheres: Cellometer Auto T4
		2.6 Imaging and Counting of Mammospheres: Celigo Image Cytometer
		2.7 Immunocytochemical Analysis of Mammospheres
		2.8 Immunolabeling
	3 Methods
		3.1 Primary Mammospheres
		3.2 Secondary Mammospheres
		3.3 Mammospheres in Matrices
		3.4 Imaging and Counting: Manual Method
		3.5 Imaging and Counting: Cellometer Auto T4
		3.6 Imaging and Counting: Celigo Image Cytometer
		3.7 Immunocytochemistry
		3.8 Immunolabeling
	4 Notes
	References
Chapter 17: Detection of Circulating and Tissue Myeloid-Derived Suppressor Cells (MDSC) by Flow Cytometry
	1 Introduction
	2 Materials
	3 Methods
		3.1 Isolation of Peripheral Blood Mononuclear Cells (PBMC)
		3.2 Mechanical and Enzymatic Dissociation for Extraction of Immune Cells from Solid Tissues
		3.3 Staining Cells with Viability Dye (See Note 9)
		3.4 Surface Markers Staining with Fluorochrome-Conjugated Antibodies
		3.5 Staining for Intracellular Arginase-1
		3.6 ROS Detection by Staining with Dihydroethidium (DHE) Indicator
		3.7 Acquisition of Data in the Flow Cytometer and Analysis (Figs. 2 and 3)
	4 Notes
	References
Chapter 18: Comet Assay for the Detection of Single and Double-Strand DNA Breaks
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Reagent Preparation
		3.2 Sample Preparation and Comet Assay
	4 Notes
	References
Chapter 19: Fluorescent-Activated Cell Sorting (Flow Cytometry)
	1 Introduction
	2 Materials
		2.1 Specimen Requirements and Criteria for Specimen Acceptability
			2.1.1 Nonacceptable Specimens
			2.1.2 Specimen Time Validation
		2.2 Equipment
		2.3 Reagents
			2.3.1 Surface Assay
			2.3.2 Cytoplasmic/Intranuclear Assay
		2.4 Cell Concentration/Antibody Optimization
		2.5 Surface Assay
		2.6 Cytoplasmic Assay
	3 Methods
		3.1 Acute Leukemia, Chronic Leukemia, and Lymphoma Panels
			3.1.1 Whole Blood and Bone Marrow
				Stain/Lyse/Wash Procedure
			3.1.2 For Tissue, FNA, CSF and All Other Body Fluids
				Lyse/Stain/Wash Procedure
			3.1.3 Procedure: Eight Color Cytoplasmic Acute Leukemia Panel
		3.2 Instrument Setup (Based on Specific Flow Cytometer)
			3.2.1 Acquisition
			3.2.2 Software Analysis Strategy
			3.2.3 Software Results Reporting
			3.2.4 Results
	4 Quality Assurance and Quality Control for Immunophenotype
	References
Index