Histochemistry of Single Molecules: Methods and Protocols

✍ Scribed by Carlo Pellicciari, Marco Biggiogera, Manuela Malatesta

• Publisher: Humana Press
• Year: 2022
• Tongue: English
• Leaves: 351
• Series: Methods in Molecular Biology, 2566
• Edition: 2
• Category: Library

✦ Synopsis

This volume details histochemical techniques for the detection of specific molecules or metabolic processes, both at light and electron microscopy. Chapters are divided into seven sections covering Vital histochemistry, Carbohydrate histochemistry, Protein histochemistry, Lipid histochemistry, Nuclear histochemistry, Plant histochemistry and Histochemistry for Nanoscience. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. The volume also contains three discursive chapters on Histochemistry in advanced cytometry, Lectins and Detection of molecules in plant cell walls by fluorescence microscopy.

 

Authoritative and cutting-edge, Histochemistry of Single Molecules: Methods and Protocols, Second Edition aims to be a useful practical guide for researchers to help further their study in this field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Histochemistry in Advanced Cytometry: From Fluorochromes to Mass Probes
1 Introduction
2 Fluorochromes and Cytometry
3 Fluorescence and Autofluorescence
4 DNA Probes
4.1 G-C Base-Specific Fluorochromes
4.2 A-T Base-Specific Fluorochromes
4.3 Intercalating Dyes
5 RNA Probes
6 Protein Probes
7 Cell Function Probes
7.1 Viability Probes
7.2 Proliferation Markers
7.3 Other Probes (for Mitochondrial Intermembrane Potential, pH, Ca++ Content, and Others)
8 Quantum Dots
9 Mass Cytometry
10 Concluding Remarks
References
Part I: Vital Histochemistry
Chapter 2: Autofluorescence Label-Free Imaging of the Liver Reticular Structure
1 Introduction
2 Materials
2.1 Samples and Chemicals
2.2 Equipment
3 Methods
3.1 Section Preparation
3.2 Imaging Microscopy
4 Notes
References
Chapter 3: Lysosome Imaging Based on Fluorescent Carbon Dots
1 Introduction
2 Materials
2.1 Chemicals, Buffers, and Cells
2.2 Equipment
3 Methods
3.1 Preparation of the CDs Stock Solution
3.2 Co-localization of CDs with LysoTracker Deep Red in Living Cells
3.3 Long-Term Staining Performance of CDs in Living Cells
3.4 Long-Term Lysosome-Targeting Ability of CDs
3.5 Lysosome Staining in Fixed Cells
4 Notes
References
Chapter 4: Oxidative and Nitrosative Stress Detection in Human Sperm Using Fluorescent Probes
1 Introduction
2 Materials
2.1 Solvents, Buffers, and Cell Media
2.2 Fluorescent Probes for ROS/RNS Detection
2.3 Controls
2.4 Equipment
3 Methods
3.1 Sperm Cells: Sample Preparation
3.2 ROS/RNS Detection
4 Notes
References
Chapter 5: Simultaneous Labeling of Adipogenic and Osteogenic Differentiating Stem Cells for Live Confocal Analysis
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Sample Labeling
2.3 Histological Validation (Optional)
2.4 Equipment
3 Methods
3.1 Cell Seeding
3.2 Cell Differentiation
3.3 Cell Labeling
3.4 Confocal Imaging
4 Notes
References
Part II: Carbohydrate Histochemistry
Chapter 6: Lectin Histochemistry: Historical Perspectives, State of the Art, and Future Directions
1 Historical Perspectives
1.1 What Are Lectins?
1.2 Lectins: A Brief Historical Introduction
2 Lectins as Tools for Histochemistry
2.1 The Development of Lectin Histochemistry
2.2 A Wider Range of Tools Become Available
2.3 Lectin Binding Carbohydrate Specificity
3 Major Applications of Lectin Histochemistry
3.1 Mapping Glycosylation Changes in Normal Human and Animal Tissues
3.2 Glycosylation Changes Associated with Progression of Malignancy Revealed by Lectin Histochemistry
4 The Next Generation of Lectin-Based Technologies
4.1 Lectins to Decipher the Complexities of the Glycome
4.2 Lectin Histochemistry Complements Other Methods of Exploration
4.3 Recombinant and Engineered Lectins
4.4 Lectin Microarrays to Explore the Glycome and Search for Glycosylation-Associated Disease Biomarkers
5 Conclusion
References
Chapter 7: Histochemical and Immunohistochemical Methods for the Identification of Proteoglycans
1 Introduction
2 Materials
2.1 Reagents for the Histochemical Methods
2.2 Reagents for Immunohistochemistry
2.3 Equipment
3 Methods
3.1 Alcian Blue pH 2.5
3.2 Safranin O
3.3 Toluidine Blue
3.4 Immunohistochemistry
4 Notes
References
Chapter 8: Combined Lectin- and Immuno-histochemistry (CLIH) for Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Paraffin Sections
2.2 Semithin Cryosections (Modified Tokuyasu Method)
2.3 CLIH
3 Methods
3.1 Paraffin Sections
3.2 Semithin Cryosections
3.3 CLIH
3.3.1 CLIH Protocol No. 1
3.3.2 CLIH Protocol No. 2
3.3.3 CLIH Protocol No. 3
3.3.4 CLIH Protocol No. 4
3.3.5 CLIH Protocol No. 5
3.4 Negative Controls
4 Notes
References
Part III: Protein Histochemistry
Chapter 9: Immunofluorescence Labeling of Skeletal Muscle in Development, Regeneration, and Disease
1 Introduction
2 Materials
2.1 Cardiac Perfusion Fixation
2.2 Immersion Fixation
2.3 Tissue Cryopreservation, Embedding, and Sectioning
2.4 Deparaffinization of Paraffin Sections (See Notes 5 and 6)
2.5 Antigen Retrieval
2.6 Immunofluorescence Staining
3 Methods
3.1 Cryopreservation of Muscle Tissue
3.1.1 Harvesting and Cryopreservation of Fresh Tissue in OCT (Fig. 2a)
3.1.2 Cardiac Perfusion Fixation and Cryopreservation of Mouse Tissue in OCT (Fig. 2b)
3.1.3 Immersion Fixation for Paraffin Embedding
3.2 Heat-Induced Antigen Retrieval
3.3 Immunofluorescence Staining
4 Notes
References
Chapter 10: Immunohistochemical Detection of the Autophagy Markers LC3 and p62/SQSTM1 in Formalin-Fixed and Paraffin-Embedded ...
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Samples
3.2 Prestaining
3.3 Chromogenic Staining
3.4 Dehydrating and Mounting Slides
3.4.1 Using Aqueous Medium
3.4.2 Using Non-aqueous Medium (See Note 12)
4 Notes
References
Chapter 11: Immunohistochemical Detection of the Chaperone-Mediated Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and P...
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Equipment
3 Methods
3.1 Preparation of Samples
3.2 Pre-staining
3.3 Chromogenic Staining
3.4 Dehydrating and Mounting Slides
3.4.1 Using Aqueous Medium
3.4.2 Using Non-aqueous Medium (See Note 12)
3.5 Evaluation of Immunohistochemical Stainings
4 Notes
References
Chapter 12: Immunogold Labeling of Milk Proteins at Transmission Electron Microscopy
1 Introduction
2 Materials
2.1 Reagents
2.2 Tools and Equipment
3 Method
3.1 Fixation and Embedding Protocol for Milk
3.2 Immunogold Labeling of β-Casein and β-Lactoglobulin
3.3 Staining of Sections
4 Notes
References
Chapter 13: Rediscover Potassium Permanganate as a Stain for Basic Proteins on Ultrathin Sections at Transmission Electron Mic...
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Equipment
3 Methods
3.1 Sample Processing
3.2 Staining
3.2.1 Staining Procedures for Chromatin Spreads
3.3 Controls
3.3.1 Negative Control
3.3.2 Positive Controls: KMnO4 Staining on Sections Immunolabeled for Basic Proteins
4 Notes
References
Part IV: Lipid Histochemistry
Chapter 14: Tissue Fixation and Processing for the Histological Identification of Lipids
1 Introduction
2 Materials
2.1 Reagents and Solutions for Tissue Fixation
2.2 Reagents and Solutions for the Freezing Technique
2.3 Reagents and Solutions for Paraffin Embedding
2.4 Equipment
3 Methods
3.1 Fixation
3.2 Freezing Technique
3.3 Paraffin Embedding
3.4 Tracy and Walia´s Post-fixation Method for Lipids
4 Notes
References
Chapter 15: Staining Methods for Normal and Regenerative Myelin in the Nervous System
1 Introduction
2 Materials
2.1 MCOLL Method
2.2 Osmium Tetroxide Method
2.3 Immunofluorescence Staining
2.4 FluoroMyelin Stain Method
2.5 Equipment
3 Methods
3.1 MCOLL Histochemical Method
3.2 Osmium Tetroxide Method
3.3 Immunofluorescence Staining Method
3.4 FluoroMyelin Stain Method
4 Notes
References
Chapter 16: Nile Red and BODIPY Staining of Lipid Droplets in Mouse Oocytes and Embryos
1 Introduction
2 Materials
2.1 Oocytes and Embryos Collection
2.2 Solutions and Material for Oocytes and Embryos Fixation
2.3 Equipment
3 Methods
3.1 Fixation
3.2 Nile Red/BODIPY Staining
3.3 Confocal Imaging and Data Analysis
4 Notes
References
Part V: Nuclear Histochemistry
Chapter 17: Chromatin Dispersion Test to Asses DNA Damage in Cervical Epithelial Cells
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Equipment
3 Methods
3.1 Specimen Preparation
3.2 Preparation of Slides with Microgel Cell Suspensions
3.3 Chromatin Dispersion Test (CDT)
3.4 Analysis
3.5 Statistics
4 Notes
References
Chapter 18: Uranyl-Free Staining as a Suitable Contrasting Technique for Nuclear Structures at Transmission Electron Microscopy
1 Introduction
2 Materials
2.1 Sample Preparation
2.1.1 Reagents
2.2 Equipment
3 Methods
3.1 Staining Procedure
3.2 Ultrastructural Immunocytochemistry
4 Notes
References
Chapter 19: Specific RNA Visualization at Electron Microscopy via Terbium Citrate Vapors
1 Introduction
2 Materials
2.1 Solutions and Embedding Media
2.2 Equipment
3 Methods
3.1 Preparation of Terbium Citrate
3.2 Sample Processing
3.3 Staining Procedure
3.4 Controls
4 Notes
References
Part VI: Plant Histochemistry
Chapter 20: Localizing Molecules in Plant Cell Walls Using Fluorescence Microscopy
1 Introduction
2 Autofluorescence of Plant Stems
2.1 Lignin
2.2 Suberin
2.3 Ferulate
2.4 Other Fluorophores
3 Immunofluorescence of Xylem Cell Walls
3.1 Embedding and Sectioning
3.2 Antibodies
3.3 Labeling Sections
4 Special Methods
4.1 En Bloc Versus Sectioning
4.2 Spectral Unmixing
4.3 Lignin Stains
4.4 Cellulose Stains
4.5 Polarized Light Imaging of Cellulose
5 Troubleshooting
6 Health and Safety Requirements
References
Chapter 21: Ratiometric Fluorescent Safranin-O Staining Allows the Quantification of Lignin Contents In Muro
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Sample Preparation
2.3 Equipment and Software
3 Methods
3.1 Plant Growth
3.2 Sample Preparation
3.3 Sample Staining
3.4 Image Capture
3.5 Image Analysis
4 Notes
References
Chapter 22: Live Fluorescence Visualization of Cellulose and Pectin in Plant Cell Walls
1 Introduction
2 Material
2.1 Material and Solutions
2.2 Equipment
3 Methods
3.1 Staining of Onion Epidermal Leaves
3.2 Staining of Arabidopsis thaliana Seedlings
3.3 Tips for Imaging Using a Confocal Laser Scanning Microscope
4 Notes
References
LiveFluorescenceVisualizationofCelluloseandPectininPlantCellWalls
Chapter 23: Staining Starch with Iodine Solution
1 Introduction
2 Materials
3 Methods
3.1 Non-embedded Sections of Maize, Wheat, and Rice Mature Seeds
3.2 LR White Resin Section of Maize Developing Seed
3.3 Isolated Starch from Maize Seeds
3.4 Staining Starch in Leaf
3.5 Staining Starch in Pollen Grain
3.6 Staining Starch in Seed of Rice
3.7 Staining Starch in Non-embedded Section of Seed
3.8 Staining Starch in Resin Section of Seed
3.9 Staining Starch Isolated from Maize Seeds
4 Notes
References
Chapter 24: Histochemical Analysis of Plant Secretory Structures
1 Introduction
2 Materials
2.1 For Detecting Hydrophilic Substances
2.2 For Detecting Lipophilic Substances, Phenolic Compounds, and Alkaloids
2.3 Extraction Solutions
2.4 Equipments
3 Methods
3.1 Detection of Hydrophilic Substances
3.1.1 Mucilage
Ruthenium Red
Alcian Blue
Tannic Acid and Ferric Chloride
3.1.2 Starch
Lugol´s Reagent
Triple Staining for Starch Detection
3.1.3 Carbohydrates
PAS (Periodic Acid-Schiff´s Reagent) Reaction
3.1.4 Callose
Aniline Blue
3.1.5 Cellulose
Calcofluor White
3.1.6 Proteins
Aniline Blue Black
Xylidine Ponceau
Coomassie Blue
3.2 Detection of Lipophilic Substances
3.2.1 Lipids (See Notes 15-18)
Sudan Black B
Sudan IV
Neutral Red
3.2.2 Cutin
Auramine O
3.2.3 Acidic and Neutral Lipids
Nile Blue (See Note 20)
3.2.4 Fatty Acids
Copper Acetate and Rubeanic Acid
3.2.5 Terpenes
NADI Reagent
3.3 Detection of Phenolic Compounds and Alkaloids
3.3.1 Phenolic Compounds
Ferric Chloride
Potassium Dichromate
Ferrous Sulfate-Formalin Fixative
3.3.2 Tannins
Vanillin-HCl
3.3.3 Lignin
Phloroglucinol-HCl
Acridine Orange
Autofluorescence
3.3.4 Alkaloids
Dragendorff´s Reagent
Wagner´s Reagent
4 Notes
References
Part VII: Histochemistry for Nanoscience
Chapter 25: Alcian Blue Staining to Visualize Intracellular Hyaluronic Acid-Based Nanoparticles
1 Introduction
2 Materials
2.1 In Vitro Cell Culture
2.2 Cell Fixation
2.2.1 Fixative Solution for Light Microscopy Analysis
2.2.2 Fixative Solution for Transmission Electron Microscopy Analysis
2.3 Alcian Blue Staining
2.4 Conventional Fluorescence Microscopy
2.5 Transmission Electron Microscopy
2.6 Equipment
3 Methods
3.1 Preparation of Cell Samples on Glass Coverslips
3.2 Cell Fixation
3.2.1 Light Microscopy
3.2.2 Transmission Electron Microscopy
3.3 Cell Sample Staining with Alcian Blue for Light and Electron Microscopy
3.4 Processing for Light Microscopy
3.5 Processing for Transmission Electron Microscopy
3.6 Controls
4 Notes
References
Chapter 26: Prussian Blue Staining to Visualize Iron Oxide Nanoparticles
1 Introduction
2 Materials
2.1 PB Staining
2.2 Staining Enhancement
2.3 Other Materials and Equipment
3 Methods
3.1 PB Staining
3.2 Interpretation
3.3 Examples of PB Staining
3.4 PB Staining Enhancement
3.4.1 Diaminobenzidine Reaction
3.4.2 Silver-Gold Intensification
4 Notes
References
Chapter 27: Diaminobenzidine Photooxidation to Visualize Fluorescent Nanoparticles in Adhering Cultured Cells at Transmission ...
1 Introduction
2 Materials
2.1 In Vitro Cell Culture
2.2 DAB Photooxidation
2.3 Transmission Electron Microscopy
2.4 Equipment
3 Methods
3.1 Preparations of Cell Samples on Glass Coverslips for DAB Photooxidation and Transmission Electron Microscopy
3.2 DAB Photooxidation
3.3 Processing for Morphological Analysis at Transmission Electron Microscopy
3.4 Controls
4 Notes
References
Chapter 28: Fluorescent Labeling of Lignin Nanocapsules with Fluorol Yellow 088
1 Introduction
2 Materials
2.1 Samples and Chemicals
2.2 Equipment
3 Methods
3.1 Preparation of Lignin Nanoparticles
3.2 Physicochemical Characterization of Empty and FY088-Loaded NCs
3.3 Fluorescence Microscope Observation
3.4 Two-Photon Observation
4 Notes
References
Index