Histology

2.

Histological Techniques and Microscopy

2.1.

Tissue Preparation

2.1.1.

Specimen Acquisition

2.1.1.1.

Biopsy Techniques

2.1.1.2.

Autopsy Procedures

2.1.1.3.

Surgical Resection

2.1.1.4.

Tissue Handling Protocols

2.1.2.

2.1.2.1.

Purpose of Fixation

2.1.2.1.1.

Prevention of Autolysis

2.1.2.1.2.

Preservation of Cellular Structure

2.1.2.1.3.

Prevention of Bacterial Decay

2.1.2.2.

Common Fixatives

2.1.2.2.1.

2.1.2.2.1.1.

Mechanism of Action

2.1.2.2.1.2.

2.1.2.2.1.3.

2.1.2.2.2.

2.1.2.2.2.1.

Use in Electron Microscopy

2.1.2.2.2.2.

Mechanism of Action

2.1.2.2.2.3.

Advantages and Limitations

2.1.2.2.3.

Mercuric Chloride Fixatives

2.1.2.2.4.

Osmium Tetroxide

2.1.2.2.5.

Alcoholic Fixatives

2.1.2.3.

Factors Affecting Fixation

2.1.2.3.1.

2.1.2.3.2.

2.1.2.3.3.

2.1.2.3.4.

2.1.2.3.5.

Fixative Volume

2.1.3.

2.1.3.1.

Purpose of Dehydration

2.1.3.2.

Dehydrating Agents

2.1.3.2.1.

2.1.3.2.2.

2.1.3.2.3.

2.1.3.3.

Graded Series Process

2.1.3.4.

Timing Considerations

2.1.4.

2.1.4.1.

Purpose of Clearing

2.1.4.2.

Clearing Agents

2.1.4.2.1.

2.1.4.2.2.

2.1.4.2.3.

2.1.4.2.4.

2.1.5.

2.1.5.1.

Purpose of Embedding

2.1.5.2.

Paraffin Wax Embedding

2.1.5.2.1.

Steps in Paraffin Embedding

2.1.5.2.2.

Temperature Control

2.1.5.2.3.

2.1.5.2.4.

2.1.5.3.

Resin Embedding

2.1.5.3.1.

2.1.5.3.2.

2.1.5.3.3.

Use in Electron Microscopy

2.1.5.4.

Frozen Section Technique

2.1.6.

2.1.6.1.

2.1.6.1.1.

Rotary Microtomes

2.1.6.1.2.

Sliding Microtomes

2.1.6.1.3.

Ultramicrotomes

2.1.6.1.4.

2.1.6.2.

2.1.6.2.1.

2.1.6.2.2.

2.1.6.2.3.

2.1.6.3.

Section Thickness

2.1.6.3.1.

Light Microscopy Sections

2.1.6.3.2.

Electron Microscopy Sections

2.1.6.3.3.

Semi-thin Sections

2.1.6.4.

Section Orientation

2.1.6.4.1.

Longitudinal Sections

2.1.6.4.2.

2.1.6.4.3.

Oblique Sections

2.1.6.4.4.

Tangential Sections

2.1.7.

Mounting on Slides

2.1.7.1.

Slide Preparation

2.1.7.2.

Adhesives and Mounting Media

2.1.7.3.

Coverslipping Techniques

2.1.7.4.

2.2.

2.2.1.

Principles of Staining

2.2.1.1.

2.2.1.2.

2.2.1.3.

2.2.1.4.

Affinity for Cellular Components

2.2.1.5.

pH Effects on Staining

2.2.2.

Hematoxylin and Eosin Staining

2.2.2.1.

2.2.2.1.1.

Nuclear Staining

2.2.2.1.2.

2.2.2.1.3.

Types of Hematoxylin

2.2.2.2.

2.2.2.2.1.

Cytoplasmic Staining

2.2.2.2.2.

Extracellular Matrix Staining

2.2.2.2.3.

2.2.2.3.

H&E Staining Protocol

2.2.2.4.

Interpretation of H&E Stains

2.2.3.

2.2.3.1.

Periodic Acid-Schiff Stain

2.2.3.1.1.

2.2.3.1.2.

2.2.3.2.

Trichrome Stains

2.2.3.2.1.

Masson's Trichrome

2.2.3.2.2.

Mallory's Trichrome

2.2.3.2.3.

Principle and Applications

2.2.3.3.

2.2.3.3.1.

Reticular Fiber Staining

2.2.3.3.2.

Nerve Fiber Staining

2.2.3.3.3.

2.2.3.4.

Elastic Fiber Stains

2.2.3.4.1.

2.2.3.4.2.

Verhoeff's Stain

2.2.3.5.

2.2.3.5.1.

2.2.3.5.2.

2.2.3.6.

2.2.3.6.1.

Prussian Blue Stain

2.2.3.7.

2.2.3.7.1.

2.2.3.7.2.

2.2.4.

Histochemistry and Cytochemistry

2.2.4.1.

Enzyme Histochemistry

2.2.4.1.1.

Alkaline Phosphatase

2.2.4.1.2.

Acid Phosphatase

2.2.4.1.3.

2.2.4.2.

Detection of Specific Molecules

2.2.4.2.1.

2.2.4.2.2.

2.2.4.2.3.

2.2.4.2.4.

2.2.5.

Immunohistochemistry

2.2.5.1.

Principle of Antibody-Antigen Binding

2.2.5.2.

Direct Immunohistochemistry

2.2.5.3.

Indirect Immunohistochemistry

2.2.5.4.

Antigen Retrieval

2.2.5.5.

Detection Systems

2.2.5.5.1.

Enzyme-Based Systems

2.2.5.5.2.

Fluorescent Systems

2.2.5.6.

Applications in Diagnosis

2.2.5.7.

Controls and Troubleshooting

2.3.

2.3.1.

The Light Microscope

2.3.1.1.

2.3.1.1.1.

2.3.1.1.2.

Objective Lenses

2.3.1.1.3.

2.3.1.1.4.

2.3.1.1.5.

2.3.1.1.6.

2.3.1.1.7.

2.3.1.1.8.

2.3.1.2.

Principles of Image Formation

2.3.1.3.

Resolution and Magnification

2.3.1.4.

Numerical Aperture

2.3.1.5.

2.3.1.6.

Limitations of Light Microscopy

2.3.1.7.

Care and Maintenance

2.3.2.

Specialized Light Microscopy

2.3.2.1.

Brightfield Microscopy

2.3.2.2.

Darkfield Microscopy

2.3.2.3.

Phase-Contrast Microscopy

2.3.2.3.1.

2.3.2.4.

Differential Interference Contrast

2.3.2.5.

Polarized Light Microscopy

2.3.2.6.

Fluorescence Microscopy

2.3.2.6.1.

2.3.2.6.2.

Excitation and Emission

2.3.2.6.3.

Filters and Light Sources

2.3.2.7.

Confocal Microscopy

2.3.2.7.1.

Principle of Optical Sectioning

2.3.2.7.2.

2.3.3.

Electron Microscopy

2.3.3.1.

Transmission Electron Microscopy

2.3.3.1.1.

2.3.3.1.2.

Sample Preparation

2.3.3.1.3.

Resolution Capabilities

2.3.3.2.

Scanning Electron Microscopy

2.3.3.2.1.

2.3.3.2.2.

Sample Preparation

2.3.3.2.3.

Three-Dimensional Imaging

2.3.3.3.

Comparison of TEM and SEM

2.3.3.4.

Limitations of Electron Microscopy

2.4.

Digital Imaging and Analysis

2.4.1.

Digital Microscopy

2.4.2.

2.4.3.

Image Processing

2.4.4.

Morphometric Analysis

2.5.

Interpreting Micrographs

2.5.1.

Understanding Planes of Section

2.5.1.1.

Longitudinal Sections

2.5.1.2.

2.5.1.3.

Oblique Sections

2.5.1.4.

Tangential Sections

2.5.2.

Identifying Common Artifacts

2.5.2.1.

Fixation Artifacts

2.5.2.2.

Processing Artifacts

2.5.2.3.

Sectioning Artifacts

2.5.2.3.1.

2.5.2.3.2.

2.5.2.3.3.

Folds and Tears

2.5.2.4.

Staining Artifacts

2.5.2.4.1.

2.5.2.4.2.

Uneven Staining

2.5.3.

Recognizing Normal Structures

2.5.4.

Identifying Pathological Changes

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1. Introduction to Histology

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3. The Cell: A Histological Perspective