Animal Models of Allergic Disease: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Applications of Mouse Models to the Study of Food Allergy
	1 Introduction
	2 Human Versus Mouse Immune System
	3 Mouse Strains
	4 Sex and  Age
	5 Use of Adjuvants
	6 Route of Exposure
	7 Microbiota
	8 Food Extract Versus Purified Proteins
	9 Summary
	References
Chapter 2: Induction of Peanut Allergy Through Inhalation of Peanut in Mice
	1 Introduction
	2 Materials
		2.1 PN Flour Suspension
		2.2 Exposing Mice to PN via Inhalation
		2.3 Crude PN Extract Suspension
		2.4 Monitoring of Anaphylaxis in  Mice
		2.5 Enzyme-Linked Immunosorbent Assay (ELISA)
	3 Methods
		3.1 Exposing Mice to PN via Inhalation: a 4-Week Model
		3.2 Biotinylation of  CPE
		3.3 ELISA for PN-Specific Antibodies
		3.4 Inducing and Monitoring Anaphylaxis to PN in  Mice
	4 Notes
	References
Chapter 3: Assessment of Immune Responses in an Animal Model of Wheat Food Allergy via Epicutaneous Sensitization
	1 Introduction
	2 Materials
		2.1 Wheat Allergen Sensitization
		2.2 Allergen-Specific IgE ELISA
		2.3 Flow Cytometry
	3 Methods
		3.1 Animal Preparation Before Sensitization on Days 0, 15, and 22
		3.2 Allergen Sensitization on Days 1, 8, 15, and 22
		3.3 ELISA for Antigen-Specific Immunoglobulin E (IgE) Quantification
		3.4 Flow Cytometry
			3.4.1 Preparation and Staining of Lymphoid Tissue Cells
			3.4.2 In Vitro Stimulation Assay
	4 Notes
	References
Chapter 4: A Mouse Model of Oral Sensitization to Hen´s Egg White
	1 Introduction
	2 Materials
		2.1 Egg White Sensitization (See Note 1)
		2.2 Egg White Challenge
		2.3 Fecal and Organ Sample Collection
		2.4 Splenocyte and MLN Cell Isolation
		2.5 Splenocyte and MLN Cell Culture
		2.6 Measurement of EW-Specific Immunoglobulins, mMCP-1, and Cytokines by ELISA
		2.7 Phenotypic Analysis of Dendritic and T Cells From MLNs and Spleens
		2.8 Intestinal Gene Expression
	3 Methods
		3.1 Egg White Sensitization and Blood Sample Collection
		3.2 Egg White Challenge and Anaphylactic Response Evaluation
		3.3 Fecal Sample Collection
		3.4 Organ Collection
		3.5 Splenocyte and MLN Cell Isolation
		3.6 Splenocyte and MLN Cell Culture
		3.7 Quantification of EW-Specific Murine IgE, IgG1 e IgG2a in Sera and IgA in Feces
		3.8 Quantification of mMCP-1 in Sera and Cytokines Levels in Supernatants of Cultured Splenocytes and MLNs
		3.9 Phenotypic Analysis of Dendritic Cells and T Cells From MLNs and Spleens
			3.9.1 Staining for Flow-Cytometric Analysis
			3.9.2 Flow-Cytometric Gating Strategy for DCs
			3.9.3 Flow-Cytometric Gating Strategy for T-Cells Subsets
		3.10 Intestinal Gene Expression
	4 Notes
	References
Chapter 5: Induction of Hypersensitivity with Purified Beta-Lactoglobulin as a Mouse Model of Cow´s Milk Allergy
	1 Introduction
	2 Materials
		2.1 Oral Sensitization
		2.2 BLG Challenge and Assessment of Allergic Reactions
	3 Methods
		3.1 Oral Sensitization
			3.1.1 Preparation of  Mice
			3.1.2 Oral Sensitization via Intragastric Gavage
		3.2 BLG Challenge and Assessment of Allergic Reactions
	4 Notes
	References
Chapter 6: Diet-Induced Mouse Model of Atopic Dermatitis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Animal Housing and Feeding
		3.2 Skin Barrier Function Measurement
		3.3 Analysis of Spontaneous Scratching
		3.4 Analysis of Ethanol-Induced Scratching
	4 Notes
	References
Chapter 7: Animal Models of Contact Dermatitis: 2,4-Dinitrofluorobenzene-Induced Contact Hypersensitivity
	1 Introduction
	2 Materials
		2.1 Sensitization of Animals
		2.2 Allergen Challenge
		2.3 Sample Collection
		2.4 Preparation of Histological Samples
		2.5 Preparation of Protein Samples
		2.6 Preparation of RNA Samples
	3 Method
		3.1 Sensitization
		3.2 Challenge and Inflammation Monitoring
		3.3 Sample Collection
		3.4 Preparation of Histological Samples
		3.5 Preparation of Protein Samples
		3.6 Preparation of RNA Samples
	4 Notes
	References
Chapter 8: Induction of Airway Hypersensitivity to Ovalbumin and Dust Mite Allergens as Mouse Models of Allergic Asthma
	1 Introduction
	2 Materials
		2.1 Allergic Asthma Mouse Models
		2.2 Pulmonary Function Test
		2.3 Examination of Bronchoalveolar Lavage (BAL) Fluid Cells
		2.4 Lung Histology
		2.5 Quantitative RT-PCR of Lung Tissue
		2.6 Lung Phenotyping by Flow Cytometry
		2.7 Total and OVA-Specific IgE Measurement
	3 Methods
		3.1 Allergic Asthma Mouse Model
			3.1.1 Sensitization and Challenge Protocol for OVA-Induced Asthma Model
			3.1.2 Challenge Protocol for HDM-Induced Asthma Model
		3.2 Pulmonary Function Test
		3.3 Examination of BAL Fluid Cells
		3.4 Lung Histology
		3.5 Th2 Cytokine Determination in the Sensitized Lung
			3.5.1 RNA Isolation and Quantitative RT-PCR of Lung Tissue
			3.5.2 Ex Vivo Lung Phenotyping by Flow Cytometry
		3.6 Total and OVA-Specific IgE Measurement
	4 Notes
	References
Chapter 9: Generation and Characterization of Inducible Lung and Skin-Specific IL-22 Transgenic Mice
	1 Introduction
	2 Materials
		2.1 Plasmids Preparation
		2.2 Generation of a TRE-Tight-IL-22 Construct
		2.3 Induction of IL-22 Expression In Vitro
		2.4 Generation of TRE-Tight-IL-22 Mice
		2.5 Histology and Immunohistochemistry
	3 Methods
		3.1 Plasmid Preparation
		3.2 Construction of a TRE-Tight-IL-22 Construct
		3.3 Induction of IL-22 Expression In Vitro
		3.4 Generation of Transgenic Mice
			3.4.1 Preparation of Mice and Zygotes
			3.4.2 Generation of TRE-Tight-IL-22 Mice
			3.4.3 Generation of Lung-Specific Inducible IL-22 Transgenic Mice
			3.4.4 Generation of Skin-Specific Inducible IL-22 Transgenic Mice
		3.5 Histology and Immunohistochemistry (IHC)
	4 Notes
	References
Chapter 10: Experimental Mouse Models of Ragweed- and Papain-Induced Allergic Conjunctivitis
	1 Introduction
	2 Materials
		2.1 RW EAC  Mouse Model
		2.2 Papain-CL Conjunctivitis Mouse Model
		2.3 Tissue Collection
		2.4 Quantitative PCR (qPCR) of the Mouse Conjunctival Tissue
		2.5 Histological Analysis of Mouse Conjunctival Tissue
		2.6 Mouse Serum IgE Quantification
		2.7 Flow Cytometric Analysis of Mouse Conjunctival Tissue for Innate Lymphoid Cell Type 2 (ILC2)
	3 Methods
		3.1 RW EAC Mouse Model
			3.1.1 Sensitization
			3.1.2 Allergen Challenge with RW Eye Drop Solution
		3.2 Papain-CL Conjunctivitis Mouse Model
		3.3 Quantitative PCR (qPCR) for Gene Expression Analysis of Th2 Cytokines
		3.4 Histological Analysis of the Mouse Conjunctiva
			3.4.1 Giemsa Staining for Eosinophil Counting
			3.4.2 Immunohistochemical Staining of the Conjunctival Tissue
		3.5 Mouse Serum IgE Quantification
		3.6 FACS Analysis of Mouse Conjunctival Tissue
			3.6.1 Cell Preparation
			3.6.2 Cell Staining
			3.6.3 Flow Cytometric Analysis
	4 Notes
	References
Chapter 11: Intravital Imaging of Vascular Permeability by Two-Photon Microscopy
	1 Introduction
	2 Materials
		2.1 Preparation of Mice
		2.2 MP Microscopy
		2.3 Tail Vein Cannula and Dye Injection
		2.4 Induction of Vascular Hyperpermeability
	3 Methods
		3.1 Preparation of Mice
		3.2 Placement of a Mouse on a MP Microscope
		3.3 Visualization of Vascular Permeability at a Homeostatic Condition
		3.4 Induction of Vascular Hyperpermeability by Histamine Injection
	4 Notes
	References
Chapter 12: Isotype-Specific Detection of Serum Immunoglobulins Against Allergens
	1 Introduction
	2 Materials
		2.1 Preparation of Serum Samples
		2.2 Antigen-Specific IgE/IgG ELISA
	3 Methods
		3.1 Preparation of Serum Samples
			3.1.1 Adsorption of IgG from Diluted Samples
			3.1.2 Elution of IgG from Magnetic Beads
		3.2 Detection of Antigen-Specific IgE or IgG with ELISA
			3.2.1 Preparation of ELISA Plate with an Antigen
			3.2.2 Colorimetric Reaction and Plate Reading
	4 Notes
	References
Chapter 13: An Overview of Flow Cytometry: Its Principles and Applications in Allergic Disease Research
	1 Introduction
	2 Principles of Flow Cytometry
	3 Designing an Antibody Panel for Flow Cytometry
		3.1 Machine Laser Configuration
		3.2 Expression Level of Antigens
		3.3 Cell Viability Marker
	4 Antibody Titration
	5 Compensation
	6 Sample Preparation
	7 Gating Strategies
	8 Controls
		8.1 Unstained Control
		8.2 Single-Stain Control
		8.3 Isotype Control
		8.4 Fluorescence-Minus-One (FMO)
	9 Applications
	References
Chapter 14: The Application of Flow Cytometry for Simultaneous and Multi-parametric Analysis of Heterogenous Cell Populations ...
	1 Introduction
	2 Materials
		2.1 Antibody Panel Design
		2.2 Preparation of Mouse Lungs
		2.3 Antibody Titration
		2.4 Cell Staining
		2.5 Acquisition and Compensation
	3 Methods
		3.1 Antibody Panel Design
		3.2 Sample Preparation of Mouse Lungs
		3.3 Antibody Titration
		3.4 Cell Staining
			3.4.1 Staining of Cell Surface Marker Proteins
			3.4.2 Intracellular Marker Staining
		3.5 Compensation
		3.6 Acquisition
		3.7 Data Analysis
	4 Notes
	References
Chapter 15: Cellular and Biochemical Analysis of Bronchoalveolar Lavage Fluid from Murine Lungs
	1 Introduction
	2 Materials
		2.1 BALF Collection
		2.2 DLC by Cytology
		2.3 DLC by Flow Cytometry
		2.4 Multiplex Cytokine Assay by Flow Cytometry
		2.5 Nitric Oxide Synthase (NOS) Assay
	3 Methods
		3.1 BALF Collection
		3.2 Total and Differential Leukocyte Counts
			3.2.1 DLC by Cytological Staining
			3.2.2 DLC by Flow Cytometry
		3.3 Multiplex Cytokine Assay by Flow Cytometry
		3.4 Nitric Oxide Synthase Activity Assay
	4 Notes
	References
Chapter 16: Procedures to Evaluate Inflammatory and Pathological Changes During Allergic Airway Inflammation
	1 Introduction
	2 Materials
		2.1 Collection and Processing of Lungs for Pathophysiological Evaluation
		2.2 Preparation of Lung Lysates
		2.3 Lung Histology
	3 Methods
		3.1 Collection of Lungs from  Mice
		3.2 Preparation of Lung Lysates
		3.3 Analysis of Th1/Th2 Cytokines and Eotaxins
		3.4 Deparaffinization and Rehydration of Tissue Sections for Histological Analyses
		3.5 Hematoxylin and Eosin (H&E) Staining for Detection of Lung Cellular Inflammation
		3.6 Periodic Acid-Schiff (PAS) Staining for Detection of Airway Mucus
		3.7 Trichrome Staining for Detection of Airway Fibrosis
		3.8 α-Smooth Muscle Actin Immunohistochemical Staining for Detection of Smooth Muscle  Mass
			3.8.1 Antigen Retrieval
			3.8.2 Quenching of Endogenous Peroxidase Activity
			3.8.3 Antibody Incubation
	4 Notes
	References
Chapter 17: Assessment of Lung Eosinophils In Situ Using Immunohistological Staining
	1 Introduction
	2 Materials
		2.1 Lung Collection for Fixation and Embedding
		2.2 Deparaffinization/Rehydration of Slides
		2.3 MBP IHC with a Red Alkaline Phosphatase Substrate as a Chromogen
		2.4 EPX IHC with DAB as a Chromogen
		2.5 EPX Fluorescent IHC with Tyramide Signal Amplification (TSA)
		2.6 EPX Indirect IF
		2.7 MBP and EPX Dual Fluorescent Immunocytochemistry (ICC)
	3 Methods
		3.1 Lung Collection for Fixation and Embedding
		3.2 Deparaffinization/Rehydration FFPE Slides
		3.3 MBP IHC with a Red AP Substrate as a Chromogen
		3.4 EPX IHC with DAB as a Chromogen
			3.4.1 Antigen Retrieval
			3.4.2 Antibody Incubation and Color Development
			3.4.3 Hematoxylin Counterstaining
			3.4.4 Dehydration and Coverslipping
		3.5 EPX Fluorescent IHC with  TSA
		3.6 EPX Indirect IF
		3.7 MBP and EPX Dual Fluorescent ICC.
	4 Notes
	References
Chapter 18: Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology
	1 Introduction
	2 Materials
		2.1 Tissue Sampling
		2.2 Tissue Processing
		2.3 H&E Staining
	3 Methods
		3.1 Tissue Sampling
		3.2 Tissue Processing
			3.2.1 Dehydration
			3.2.2 Paraffin Embedding
			3.2.3 Slide Preparation
		3.3 H&E Staining
	4 Notes
	References
Chapter 19: Antibiotic Treatment in an Animal Model of Inflammatory Lung Disease
	1 Introduction
	2 Materials
		2.1 Antibiotic Treatment
		2.2 Papain-Induced Asthma Model
		2.3 Collection of Bronchiolar Lavage (BAL) Fluid, Lungs, and Blood Samples
		2.4 Histology
		2.5 Reverse-Transcription Quantitative PCR (qPCR)
		2.6 Assessment of IgE Levels in Serum Samples
		2.7 Flow Cytometry
	3 Methods
		3.1 Antibiotic Treatment
		3.2 Papain-Induced Asthma Model
		3.3 Collection of Bronchiolar Lavage (BAL) Fluid, Lungs, and Blood
		3.4 Histology
		3.5 Reverse-Transcription  qPCR
			3.5.1 RNA Isolation from Lung Tissue
			3.5.2 Reverse-Transcription
			3.5.3 Real-Time Quantitative  PCR
		3.6 Assessment of IgE Levels in Serum Samples
		3.7 Flow Cytometry
	4 Notes
	References
Chapter 20: Methods for Experimental Allergen Immunotherapy: Subcutaneous and Sublingual Desensitization in Mouse Models of Al...
	1 Introduction
	2 Materials
		2.1 Subcutaneous and Sublingual Immunotherapy in a Mouse Model of Allergic Asthma
		2.2 Blood Withdrawal via Orbital Puncture
		2.3 Ear Swelling Test
		2.4 Lung Function Measurement
		2.5 Analysis of the Infiltration of Inflammatory Cells in BALF
		2.6 Preparation of Single-Cell Suspensions of Lung Tissue, Spleen, and Draining Lymph Nodes (DLNs)
		2.7 Restimulation of Lung Cells and Draining Lymph Node (DLN) Cells
		2.8 Quantification of Lung Single-Cell Suspensions Using Flow Cytometry
		2.9 Homogenization of Lung Tissue for Total Protein and Cytokine Analysis
		2.10 Biotinylation of Allergens for spIgE ELISA
		2.11 Analysis of Immunoglobulin Levels in Serum with ELISA
		2.12 Analysis of Cytokine Levels in BALF, Supernatant of Restimulated Single-Cell Suspensions and Lung Tissue Homogenates
	3 Methods
		3.1 Sensitization
		3.2 SCIT Treatments
			3.2.1 SCIT Treatment of BALB/cByJ Mice
			3.2.2 SCIT Treatment of C57BL/6 Mice
		3.3 SLIT Treatments
		3.4 Allergen Challenges
		3.5 Blood Withdrawal via Retro-Orbital Puncture
		3.6 Ear Swelling Test
		3.7 Lung Function Measurement
			3.7.1 Preparation
			3.7.2 Methacholine Challenge
		3.8 Collection of Blood, Bronchoalveolar Lavage, and Lung Tissue
		3.9 Analysis of the Infiltration of Inflammatory Cells in BALF
			3.9.1 Cytological Analysis with Cytospin Preparations
		3.10 Preparation of Single-Cell Suspensions of Lung Tissue, DLNs, and the Spleen
			3.10.1 Single-Cell Suspensions from Lung Tissue and DLNs
			3.10.2 Single-Cell Suspensions from the Spleen
		3.11 Restimulation of Lung Cells and DLN Cells
		3.12 Quantification of DCs, T-Cell Populations, and Innate Lymphoid Cells in Lung Single-Cell Suspensions Using Flow Cytometry
			3.12.1 Staining of Extracellular and Intracellular Targets
			3.12.2 Flow Cytometry
		3.13 Homogenization of Lung Tissue for Total Protein and Cytokine Analysis
		3.14 Analysis of Immune Responses by ELISA
			3.14.1 Biotinylation of Allergens for spIgE ELISA
			3.14.2 Detection of Analytes in Serum, BALF, Culture Media, and Tissue Homogenates by ELISA
	4 Notes
	References
Chapter 21: T-Cell Epitope Immunotherapy in Mouse Models of Food Allergy
	1 Introduction
	2 Materials
		2.1 Allergen Sensitization and T-Cell Epitope Immunotherapy
		2.2 Allergen Challenge
		2.3 ELISA for Allergen-Specific Antibodies
		2.4 Mouse Splenocytes Preparation
		2.5 Intestinal Preparation for Histological Analysis
		2.6 Immunohistochemical Staining for Eosinophils and Foxp3+ Cells
	3 Methods
		3.1 Allergen Sensitization
		3.2 T-Cell Epitope Treatment
		3.3 Allergen Challenges
			3.3.1 Preparation and Administration of Culprit Allergens
			3.3.2 Assessment of Immediate Allergic Responses
			3.3.3 Tail Vein Blood Sampling
		3.4 ELISA for Allergen-Specific Antibodies
		3.5 Mouse Splenocytes Preparation
		3.6 Intestinal Preparation for Histological Analysis
		3.7 Immunohistochemical Staining for Eosinophils and Foxp3+ Cells
	4 Notes
	References
Index