Front Cover

The Handbook of Histopathological Practices in Aquatic Environments

Copyright Page

Contents

Foreword

Preface

Acknowledgments

List of Abbreviations and Acronyms

1 Introduction

1.1 A Brief Account on the History and Purpose of Histology and Histopathology in Aquatic Organisms

1.2 Histopathological Traits as Biomarkers in Marine Environmental Science

1.3 Target Species and Organ: The Importance of Understanding Toxicological Pathways

1.4 Histopathology Links Many Levels of Biological Organization: The Systems Biology Approach

1.5 How to Use This Book

2 Fundamental Concepts

2.1 Procedural Overview

2.2 Basic Equipment and Material

2.2.1 Essential Equipment

2.2.2 Glass and Plasticware for Sample Preparation

2.2.3 Microscope slides, Coverslips, and Adhesives

2.2.4 Microtome Knives and Blades

2.3 Important Considerations on Solvents and Reagents

2.3.1 Fixatives

2.3.2 Embedding Media

2.3.3 Histological Dyes

2.3.4 Waters

2.3.5 Alcohols and Other Solvents

2.3.6 Buffer Systems

2.3.7 Mounting Media

2.4 Safety Precautions

3 Sample Preparation

3.1 Tissue Preparation and Fixation

3.1.1 Harvesting Material

3.1.2 Fixation

3.1.3 Additional Treatments

3.2 Paraffin-Embedded Samples

3.2.1 General Procedure for Paraffin Infiltration and Embedding

3.2.2 Casting Paraffin Blocks

3.2.3 Sectioning Paraffin Sections With a Rotary Microtome

3.2.4 Staining Paraffin-Embedded Samples

3.3 Procedures for Resin Embedding

3.3.1 Embedding Samples in Resins

3.3.2 Obtaining Semithin and Thin Section From Resin-Embedded Samples

3.3.3 Staining and Contrasting Resin Sections

4 Staining Protocols

4.1 General Histological Stains

4.1.1 Hematoxylin and Eosin

4.1.1.1 Introduction

4.1.1.2 Procedure

4.1.2 Toluidine Blue

4.1.2.1 Introduction

4.1.2.2 Procedure

4.2 Histochemistry of Lipids and Sugars

4.2.1 Alcian Blue/Nuclear Fast Red Stain for Acidic Polysaccharides

4.2.1.1 Introduction

4.2.1.2 Procedure

4.2.2 Lipid Staining With Osmium Tetroxide

4.2.2.1 Introduction

4.2.2.2 Procedure

4.2.3 Periodic Acid/Schiff’s Stain for Polysaccharides

4.2.3.1 Introduction

4.2.3.2 Procedure

4.2.4 Sudan Black for Lipids

4.2.4.1 Introduction

4.2.4.2 Procedure

4.3 Peptide Histochemistry

4.3.1 Coomassie Blue Stain for Proteins

4.3.1.1 Introduction

4.3.1.2 Procedure

4.4 Histochemistry of Metals, Metalloids, and Metallic Compounds

4.4.1 Some Important Considerations on Metal Histochemistry

4.4.2 Castel’s Method for Arsenic

4.4.2.1 Introduction

4.4.2.2 Procedure

4.4.3 Perl’s Prussian Blue for Hemosiderin

4.4.3.1 Introduction

4.4.3.2 Procedure

4.4.4 Rubeanic Acid Method for Copper

4.4.4.1 Introduction

4.4.4.2 Procedure

4.4.5 von Kossa’s Stain for Calcium

4.4.5.1 Introduction

4.4.5.2 Procedure

4.5 Stains for Microorganisms

4.5.1 Gram’s Stain for Paraffin Sections

4.5.1.1 Introduction

4.5.1.2 Procedure

4.5.2 Staining Acid-Fast Microorganisms

4.5.2.1 Introduction

4.5.2.2 Procedure

4.6 Polychrome Stains

4.6.1 Masson’s Trichrome

4.6.1.1 Introduction

4.6.1.2 Procedure

4.6.2 Tetrachrome Stain

4.6.2.1 Introduction

4.6.2.2 Procedure

4.6.3 van Gieson’s Trichrome (Elastic Stain)

4.6.3.1 Introduction

4.6.3.2 Procedure

4.7 Techniques for Fluorescence Microscopy

4.7.1 Acridine Orange Stain for Nucleic Acids

4.7.1.1 Introduction

4.7.1.2 Procedure

4.7.2 DAPI Nuclear Stain

4.7.2.1 Procedure

4.8 Immunohistochemistry

4.8.1 Principle and Purpose

4.8.2 Sample Preparation

4.8.3 Choosing Primary and Secondary Antibodies

4.8.4 General Protocol for Immunohistochemistry for Bright-Field and Fluorescence Microscopy

5 Microphotography and Image Processing: Creating Artwork

5.1 Background

5.2 General Considerations on Digital Images and File Types

5.3 Image Resolution and Print Size

5.4 Color and Grayscale

5.5 Correcting Color and Shading

5.6 Post Hoc Image Corrections

6 Identification of Major Histopathological Traits

6.1 Understanding Deviations From Normal Histological Features in Aquatic Animals

6.1.1 Some Notes on Basic Tissue Types

6.1.2 Normal Structure of the Liver and Its Analogues

6.1.3 Normal Structure of Gills

6.1.4 Reproductive Organs

6.2 Inflammation and Circulatory Disorders

6.3 Diagnosing Cell Death

6.4 Degenerative Disorders

6.5 Regressive Alterations

6.6 Cell Proliferation

6.7 Preneoplasms and Neoplasms

6.8 Symbionts, Parasites, and Commensals

7 Scoring and Data Processing

7.1 Fundamental Principles of Histopathological Scoring

7.2 Quantitative Histopathology

7.3 Semiquantitative Histopathology

7.4 Experimental Design and Statistical Processing of Data

7.4.1 Bioassays and Passive Biomonitoring

7.4.2 Types of Variables

7.4.3 Number of Individuals and Replication

7.4.4 Basic Statistical Approaches

8 Common Problems and Troubleshooting

8.1 Histological Artifacts and Misdiagnosis

8.1.1 Inflammation, Circulation, and Related Disorders

8.1.2 Misdiagnosing Cell Death

8.1.3 Gill Processing Artifacts and Misdiagnosis

8.1.4 Misdiagnosing Progressive Disorders

8.2 Technical Problems in Sample Processing

8.2.1 Troubleshooting

8.2.2 Solutions for Sectioning Bone and Other Highly Calcified Samples

Appendix A: Fixative Solutions

A.1 Bouin’s Solution

A.2 Carnoy’s Fluid

A.3 Castel’s Fixative

A.4 Davidson’s Fixative

A.5 Glutaraldehyde Fixative (Standard)

A.6 Neutral-Buffered Formalin

A.7 Osmium Tetroxide

A.8 Paraformaldehyde Fixative (PFA)

A.9 Zenker’s Fixative

Appendix B: Dyes

B.1 Alcian Blue

B.2 Aniline Blue

B.3 Aqueous Picric Acid

B.4 Biebrich’s Scarlet–Acid Fuchsin

B.5 Carbol Fuchsin

B.6 Crystal Violet (Hucker–Conn’s)

B.7 DAPI Nuclear Stain

B.8 Eosin (Alcoholic)

B.9 Fisher’s Coomassie Blue

B.10 Hematoxylin (Alum–Based)

B.11 Methylene Blue

B.12 Neutral Red

B.13 Nuclear Fast Red

B.14 Paragon

B.15 Reynolds’ Lead Citrate

B.16 Rubeanic Acid

B.17 Schiff’s Reagent (for Periodic Acid–Schiff’s Procedure)

B.18 Toluidine Blue

B.19 Uranyl Acetate

B.20 Weigert’s Iodine

B.21 Weigert’s Iron Hematoxylin

B.22 van Gieson’s Dye

Appendix C: Accessory Solutions

C.1 Acid Alcohol

C.2 Cacodylate Buffer

C.3 Gum Arabic, Mounting Medium

C.4 Histological Adhesive

C.5 Phosphate Buffer (PB) and Phosphate-Buffered Saline (PBS)

C.6 Phosphotungstic/Phosphomolybdic Acid

C.7 Scott’s Tap Water Substitute

C.8 Sodium Ethoxide or Methoxide (Epon Removal)

C.9 Trypsin Solution

Appendix D: Rapid Protocols

D.1 Hematoxylin & Eosin Staining

D.1.1 Start Notes

D.1.2 Procedure

D.2 Rapid Protocol for Fluorescence Immunohistochemistry

D.2.1 Start Notes

D.2.2 Procedure

D.3 Basic Preparation of Samples for TEM

D.3.1 Start Notes

D.3.2 Procedure

Appendix E: Accessory Tables

E.1 Gay–Lussac’s Table for Ethanol Dilutions

E.2 Ultramicrotome Section Thickness Color Reference Chart

References

Index

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