The Olfactory System (Methods in Molecular Biology, 2710)

Preface
Contents
Contributors
Chapter 1: Visualizing and Manipulating Olfactory Cilia Through Viral Delivery Coupled with En Face Imaging of Intact OE
	1 Introduction
	2 Materials
		2.1 Intranasal Viral Administration
		2.2 Dissection
		2.3 Confocal Microscopy and Analysis
	3 Methods
		3.1 Intranasal Viral Delivery
		3.2 Tissue Preparation
		3.3 En Face Imaging
			3.3.1 Static Condition Imaging
			3.3.2 TIRF Imaging
			3.3.3 FRAP Imaging
		3.4 Image Processing and Data Analysis
			3.4.1 Static Condition Analysis
			3.4.2 TIRF Analysis
			3.4.3 FRAP Analysis
	4 Notes
	References
Chapter 2: Genome-Wide RNA Tomography in the Mouse Whole Olfactory Mucosa
	1 Introduction
	2 Materials
		2.1 WOM Dissection and Cryosection
		2.2 RNA Extraction from WOM Cryosections
		2.3 cDNA Synthesis and Amplification and RNA-seq Library Preparation
	3 Methods
		3.1 WOM Dissection and Cryosection
		3.2 RNA Extraction from WOM Cryosections
		3.3 cDNA Synthesis and Amplification
		3.4 Preparation of RNA-Sequencing Libraries
	4 Notes
	References
Chapter 3: Optical Activation of Photoswitchable TRPC Ligands in the Mammalian Olfactory System Using Laser Scanning Confocal ...
	1 Introduction
	2 Materials
		2.1 Preparation of Physiological Solutions
		2.2 Preparation of Photoswitchable Ligands
		2.3 Preparation of Ca2+-Indicator Dye Solution
		2.4 Preparation of Tissue Slice Transfer Pipette
	3 Methods
		3.1 Optical Activation of Photoswitchable TRPC Ligands Using Laser Scanning Confocal Microscopy
	4 Notes
	References
Chapter 4: Intranasal Pressure Recording for Monitoring Mouse Respiration
	1 Introduction
	2 Materials
		2.1 Surgery Equipment
		2.2 Animal Care: Pre- and Postoperative Needs
		2.3 Recording Equipment and Analysis
	3 Methods
		3.1 Preoperative Care
		3.2 Operation
		3.3 Postoperative Care
		3.4 Recording and Analysis
	4 Notes
	References
Chapter 5: Identification of Immune Cells in Murine Olfactory Mucosa
	1 Introduction
	2 Materials
		2.1 Generation of Olfactory Mucosa Single-Cell Suspensions for Flow Cytometry
		2.2 Immunofluorescent Histochemistry of Olfactory Mucosa
		2.3 Intravenous Antibody labeling
	3 Methods
		3.1 Generation of Olfactory Mucosa Single-Cell Suspensions for Flow Cytometry
		3.2 Immunofluorescent Histochemistry of Olfactory Mucosa
		3.3 Intravenous Antibody Labeling
	4 Notes
	References
Chapter 6: Chromatin Immunoprecipitation from Formaldehyde Cross-Linked Olfactory Sensory Neurons
	1 Introduction
	2 Materials
		2.1 Isolation and Fixation of Dissociated Cells
		2.2 Cell Lysis and Chromatin Sonication
		2.3 Chromatin Immunoprecipitation
	3 Methods
		3.1 Isolation and Fixation of Dissociated Cells
		3.2 Cell Lysis and Sonication
		3.3 Chromatin Immunoprecipitation
	4 Notes
	References
Chapter 7: Activity-Dependent Labeling of Olfactory Sensory Neurons Using RNA Fluorescence In Situ Hybridization Followed by P...
	1 Introduction
	2 Materials
		2.1 OE Sections
		2.2 OR Probe Preparation
			2.2.1 PCR Amplification of DNA Template
			2.2.2 Riboprobe Synthesis
		2.3 RNA FISH
			2.3.1 Pre-hybridization and Hybridization
			2.3.2 Post-hybridization Washes and Fluorescent Detection Using TSA
		2.4 pS6 IF
	3 Methods
		3.1 OE Sections (Fig. 2)
		3.2 OR Probe Preparation
			3.2.1 PCR Amplification of DNA Template
			3.2.2 Probe Synthesis, Hydrolysis, and Cleanup
		3.3 RNA FISH
			3.3.1 Pre-hybridization (Day 1)
			3.3.2 Hybridization (Day 1)
			3.3.3 Post-Hybridization Washes and Fluorescent Detection Using TSA (Day 2)
		3.4 pS6 IF (Days 2 and 3)
	4 Notes
	References
Chapter 8: Measuring Cell Surface Expression of Odorant Receptors via Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Equipment and Software
		2.2 Reagents and Labware
	3 Methods
		3.1 Plate Seeding
		3.2 Transfection
		3.3 Sample Preparation
		3.4 Flow Cytometry Gating Setup
		3.5 Sample Reading
		3.6 Analysis
	4 Notes
	References
Chapter 9: Dissociation of Mouse Olfactory Mucosae for Fluorescence-Activated Cell Sorting of Olfactory Sensory Neurons
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Enzymes
		2.3 Sorting
	3 Methods
		3.1 Preparations
		3.2 Dissociation of Olfactory Mucosae
		3.3 Optimization, Calibration, and Standardization of the Cell Sorter
	4 Notes
	References
Chapter 10: Preparation of Human Olfactory Epithelial Biopsies for Downstream Analysis
	1 Introduction
	2 Materials
		2.1 Instruments/Supplies for Collecting Surgical Olfactory Mucosa Biopsy
		2.2 Instruments/Supplies for Collecting Cytology Biopsy
		2.3 Tissue Dissociation
		2.4 Processing for Frozen Section Immunohistochemistry
		2.5 Instruments or Kits for Potential Downstream Assays
	3 Methods
		3.1 Obtaining Surgical Biopsies of the Human Olfactory Mucosa
		3.2 Nasal Cytology Brush Biopsies of the Human Olfactory Mucosa
		3.3 Tissue Processing for Frozen Section Immunohistochemistry
		3.4 Sample Processing for Single-Cell Sequencing
	4 Notes
	References
Chapter 11: Cranial Window for Acute and Chronic Optical Access to Record Neuronal Network Dynamics in the Olfactory Bulb
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Solutions and Drugs
		2.3 Equipment
		2.4 Surgical Instruments and Various Items
	3 Methods
		3.1 Procedures for Injecting AAV Expressing Genetically Encoded Sensors into the Olfactory Bulb
		3.2 Surgical Procedures for Viral Injections
		3.3 AAV Injections
		3.4 Procedures for Cranial Window Implantation for Acute Imaging in Anesthetized Mice
		3.5 Procedures for Chronic Cranial Window Implantation for Chronic Imaging
	References
Chapter 12: Target-Captured mRNA from Murine Olfactory Bulb
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Solutions
	3 Methods
		3.1 Olfactory Bulb Sectioning
			3.1.1 Vibratome Setup and Experimental Preparation
			3.1.2 Subjects
			3.1.3 Olfactory Bulb Dissection and Embedding
			3.1.4 Olfactory Bulb Sectioning
		3.2 RNA Extraction
			3.2.1 Tissue Homogenization
			3.2.2 RNA Extraction
			3.2.3 RNA Quantification
		3.3 cDNA Synthesis
			3.3.1 First-Strand cDNA Synthesis
			3.3.2 cDNA Amplification
			3.3.3 cDNA Purification
		3.4 DNA Sequencing Library Construction
			3.4.1 cDNA Fragmentation
			3.4.2 End Repair and A-Tailing
			3.4.3 Adapter Ligation
			3.4.4 Bead Purification of Adapter-Ligated Library
			3.4.5 Library Amplification
			3.4.6 Bead Purification of Amplified Library
		3.5 Enrichment of Target Sequences
			3.5.1 Probe Hybridization
			3.5.2 Target Transcript Recovery
			3.5.3 Ligation-Mediated (LM) PCR Amplification
			3.5.4 LM-PCR Purification
	References
Chapter 13: Identification and Localization of Cell Types in the Mouse Olfactory Bulb Using Slide-SeqV2
	1 Introduction
	2 Materials
	3 Methods
		3.1 Tissue Dissection and Embedding
		3.2 Tissue Sectioning
		3.3 Library Preparation
		3.4 Sequencing
		3.5 Sequencing Data Analysis
	4 Notes
	References
Chapter 14: Dense and Sparse Labelling of Mitral Cells by Oral and Intraperitoneal Routes of Tamoxifen Administration
	1 Introduction
	2 Materials
	3 Method
		3.1 Dense Labelling with Tamoxifen-Containing Chow
			3.1.1 Day 1: Replacing the Normal Food with Tamoxifen-Containing Food
			3.1.2 Days 2-3: Oral tamoxifen Administration Via Tamoxifen-Containing Diet
			3.1.3 Day 4: Post-administration Monitoring and Determination of tamoxifen Consumed by Group-Housed Mice
			3.1.4 Day 14 Onward: Induced Expression Level Should Be Suitable for Histology or Other Experiments (Fig. 2)
		3.2 Sparse Labelling with an Intraperitoneal Injection
			3.2.1 Day 1: Intraperitoneal Injection of Tamoxifen
			3.2.2 Day 2: Monitoring
			3.2.3 Day 14 Onward: Induced Expression Level Should Be Suitable for Histology or Other Experiments (Fig. 2)
	4 Notes
	References
Chapter 15: Techniques in Staining of Rodent and Human Olfactory Tissue
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 16: Electrophysiological Recordings from Identified Cell Types in the Olfactory Cortex of Awake Mice
	1 Introduction
	2 Materials and Methods
	3 Notes
	References
Correction to: Identification and Localization of Cell Types in the Mouse Olfactory Bulb Using Slide-SeqV2
Index