Ross Histology Text and Atlas 6th(1)

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HISTOLOGYA Text and Atlas

with Correlated Cell and Molecular Biology

Sixth Edition

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Michael H. Ross (19302009)

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Michael H. Ross, PhD (deceased)Professor and Chairman EmeritusDepartment of Anatomy and Cell BiologyUniversity of Florida College of MedicineGainesville, Florida

Wojciech Pawlina, MDProfessor and Chair Department of AnatomyDepartment of Obstetrics and GynecologyAssistant Dean for Curriculum Development and InnovationMayo Medical School College of Medicine, Mayo Clinic Rochester, Minnesota

with Correlated Cell and Molecular Biology

Sixth Edition

HISTOLOGYA Text and Atlas

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Acquisitions Editor: Crystal TaylorProduct Manager: Jennifer VerbiarDesigner: Doug SmockCompositor: MPS Limited, A Macmillan Company

Sixth Edition

Copyright 2011 Lippincott Williams & Wilkins, a Wolters Kluwer business.

Two Commerce Square351 West Camden Street 2001 Market StreetBaltimore, MD 21201 Philadelphia, PA 19103

All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, includ-ing as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permissionfrom the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individu-als as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please con-tact Lippincott Williams & Wilkins at 530 Walnut Street, Philadelphia, PA 19106, via email at [email protected], or via website at lww.com(products and services).

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Library of Congress Cataloging-in-Publication Data

Ross, Michael H.Histology: a text and atlas: with correlated cell and molecular biology/Michael H. Ross, Wojciech Pawlina.6th ed.

p. ; cmIncludes bibliographical references and index.ISBN 978-0-7817-7200-6 (alk. paper)1. Histology. 2. HistologyAtlases. I. Pawlina, Wojciech. II. Title. [DNLM: 1. HistologyAtlases. QS 517 R825h 2011]QM551.R67 2011611.018dc22

2010024700

DISCLAIMERCare has been taken to confirm the accuracy of the information present and to describe generally accepted practices. However, the authors, editors,and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no war-ranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. Application of this informationin a particular situation remains the professional responsibility of the practitioner; the clinical treatments described and recommended may not beconsidered absolute and universal recommendations.

The authors, editors, and publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accordance with thecurrent recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and theconstant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changein indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new or infre-quently employed drug.

Some drugs and medical devices presented in this publication have Food and Drug Administration (FDA) clearance for limited use in restricted re-search settings. It is the responsibility of the health care provider to ascertain the FDA status of each drug or device planned for use in their clinicalpractice.

To purchase additional copies of this book, call our customer service department at (800) 638-3030 or fax orders to (301) 223-2320. Internationalcustomers should call (301) 223-2300.

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This edition is dedicated to my wife Teresa Pawlina whose love, patience, and endurance created safe havens for working on this project and to my children Conrad Pawlina and Stephanie Pawlina

whose stimulation and excitement have always kept my catecholamine levels high.

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This sixth edition of Histology: A Text and Atlas with CorrelatedCell and Molecular Biology continues a tradition of providingmedical, dental, and allied health science students with a tex-tual and visual introduction to histology with correlative cellbiology. As in previous editions, this book is a combinationtext-atlas in that standard textbook descriptions of histologicprinciples are supplemented by illustrations and photographs.In addition, separate atlas sections follow each chapter and pro-vide large-format, labeled atlas plates with detailed legendshighlighting elements of microanatomy. Histology: A Text andAtlas is therefore two books in one.

Significant modifications have been made in this editionin order to create an even more useful and understandable ap-proach to the material:

Updated cellular and molecular biology. Material intro-duced in the fifth edition has been updated to include the lat-est advancements in cellular and molecular biology. The sixthedition focuses on selected information to help students withoverall comprehension of the subject matter. To accommo-date reviewers suggestions, the sixth edition also integratesnew cell biology information into several chapters. For in-stance, the cell biology of endothelial cells has been added tothe discussion of the cardiovascular system; a section on pri-mary cilia, including their structure and function, was addedto the epithelial tissue chapter; a new clinical nomenclaturefor cells involved in hemopoiesis and a detailed description ofthe respiratory burst reaction in neutrophils were added tothe chapter on blood; new information and diagrams of nervefiber regeneration were added to the nerve tissue chapter; andthe cell biology of taste receptors was incorporated into thechapter on the digestive system.

Reader-friendly innovations. The book has been redesignedin an attempt to provide more ready access to important con-cepts and essential information. Additional color font is usedin the body of the text. Important concepts are listed as sen-tence headings. Features of cells, tissues, and organs and theirfunctions, locations, and other relevant short phrases are for-matted as bulleted lists that are clearly identifiable in the bodyof the text by oversized color bullets. Essential terms within each

specific section are introduced in the text in an eye-catchingoversized red bolded font that clearly stands out from the re-maining black text. Text containing clinical information or thelatest research findings is presented in blue, with terminologypertaining to diseases, conditions, symptoms, or causativemechanisms in oversized bolded blue. The clinical sections ofthe text are easily found within each chapter.

Emphasis on features. Many of the pedagogic featuresfrom the last edition have been refined, and some new fea-tures have been added:

More summary tables are included to aid students in lear -ning and reviewing material without having to rely onstrict memorization of data. These include a review table ofthe specializations in the apical domains of epithelial cellsand a table of features of adipose tissue. Many tables havebeen updated and modified.

Previous clinical and functional correlations boxes havebeen replaced with Clinical Correlation and FunctionalConsideration Folders. More new folders have been addedto each chapter, and existing folders have been redesigned,updated, enhanced, and illustrated with new diagrams andimages of clinical specimens. New folders contain clinicalinformation related to the symptoms, photomicrographsof diseased tissues or organs, short histopathological descriptions, and treatment of specific diseases. Importantterms have been highlighted with oversized bolded text.While the information in these folders might be considered ancillary material, it demonstrates the functional impact and clinical significance of histology.

More Atlas Plates have been added to the atlas section atthe end of each chapter. Several orientation micrographswere added to the summary box in the atlas section. Atlasplates for the blood chapters have been completely re-designed so as to show both mature forms of blood cellsand the stages through which they pass duringhemopoiesis. Many plates have been replaced with vibrantdigital images.

More new figures and illustrations have also been added,and about one-third of all old figures have been redrawn for

Preface

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greater clarity and conceptual focus. This sixth edition in-corporates many new clinical images and photomicrographsto illustrate information in the clinical correlation folders.Many new high-resolution digital photomicrographs havebeen integrated into each chapter.

New design. A bright, energetic text design sets off the newillustrations and photos and makes navigation of the texteven easier than in previous editions.

As in the last five editions, all of the changes were under-taken with student needs in mind; namely, to understand thesubject matter, to become familiar with the latest informa-tion, and to be able to practically apply newfound knowledge.

Wojciech Pawlina

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This sixth edition of Histology: A Text and Atlas with Corre-lated Cell and Molecular Biology reflects continued improve-ment on previous editions. The changes that have been madecome largely from comments and suggestions by studentswho have taken the time and effort to tell us what they likeabout the book and, more importantly, how it might be im-proved to help them better understand the subject matter.The majority of such comments and suggestions have beenincorporated into this new edition.

Many of our colleagues who teach histology and cell biol-ogy courses were likewise most helpful in creating this newedition. Many of them suggested a stronger emphasis on clin-ical relevance, which we responded to as best we could withinpage limitations. Others were most helpful in providing newmicrographs, suggesting new tables, and redrawing existingdiagrams and figures.

Specifically, we owe our thanks to the following reviewers,both students and faculty, who spent considerable time andeffort to provide us with corrections and suggestions for im-provement. Their comments were a valuable source of infor-mation in planning this sixth edition.

Irwin Beitch, PhDQuinnipiac UniversityHamden, Connecticut

Paul B. Bell, Jr., PhDUniversity of OklahomaNorman, Oklahoma

David E. Birk, PhDUniversity of South Florida, College of MedicineTampa, Florida

Christy Bridges, PhDMercer University School of MedicineMacon, Georgia

Benjamin S. Bryner, MDUniversity of Michigan Medical SchoolAnn Arbor, Michigan

Craig A. Canby, PhDDes Moines UniversityDes Moines, Iowa

Stephen W. Carmichael, PhDCollege of Medicine, Mayo ClinicRochester, Minnesota

John Clancy, Jr., PhDLoyola University Medical CenterMaywood, Illinois

Rita Colella, PhDUniversity of Louisville School of MedicineLouisville, Kentucky

Iris M. Cook, PhDState University of New York Westchester Community CollegeValhalla, New York

Jolanta Durski, MDCollege of Medicine, Mayo ClinicRochester, Minnesota

William D. Edwards, MDCollege of Medicine, Mayo ClinicRochester, Minnesota

Bruce E. Felgenhauer, PhDUniversity of Louisiana at LafayetteLafayette, Louisiana

Amos Gona, PhDUniversity of Medicine & Dentistry of New JerseyNewark, New Jersey

Ervin M. Gore, PhDMiddle Tennessee State UniversityMurfreesboro, Tennessee

Acknowledgments

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Joseph P. Grande, MD, PhDCollege of Medicine, Mayo ClinicRochester, Minnesota

Joseph A. Grasso, PhDUniversity of Connecticut Health CenterFarmington, Connecticut

Jeremy K. Gregory, MDCollege of Medicine, Mayo ClinicRochester, Minnesota

Brian H. Hallas, PhDNew York Institute of TechnologyOld Westbury, New York

Charlene Hoegler, PhDPace UniversityPleasantville, New York

Cynthia J. M. Kane, PhDUniversity of Arkansas for Medical SciencesLittle Rock, Arkansas

Thomas S. King, PhDUniversity of Texas Health Science Center at San AntonioSan Antonio, Texas

Penprapa S. Klinkhachorn, PhDWest Virginia UniversityMorgantown, West Virginia

Bruce M. Koeppen, MD, PhDUniversity of Connecticut Health CenterFarmington, Connecticut

Beverley Kramer, PhDUniversity of the WitwatersrandJohannesburg, South Africa

Craig Kuehn, PhDWestern University of Health SciencesPomona, California

Nirusha Lachman, PhDCollege of Medicine, Mayo ClinicRochester, Minnesota

Priti S. Lacy, PhDDes Moines University, College of Osteopathic MedicineDes Moines, Iowa

H. Wayne Lambert, PhDWest Virginia UniversityMorgantown, West Virginia

Gavin R. Lawson, PhDWestern University of Health SciencesBridgewater, Virginia

Susan LeDoux, PhDUniversity of South AlabamaMobile, Alabama

Karen Leong, MDDrexel University College of MedicinePhiladelphia, Pennsylvania

A. Malia Lewis, PhDLoma Linda UniversityLoma Linda, California

Wilma L. Lingle, PhDCollege of Medicine, Mayo ClinicRochester, Minnesota

Frank Liuzzi, PhDLake Erie College of Osteopathic MedicineBradenton, Florida

Donald J. Lowrie, Jr., PhDUniversity of Cincinnati College of MedicineCincinnati, Ohio

Andrew T. Mariassy, PhDNova Southeastern University College of Medical SciencesFort Lauderdale, Florida

Geoffrey W. McAuliffe, PhDRobert Wood Johnson Medical SchoolPiscataway, New Jersey

Kevin J. McCarthy, PhDLouisiana State University Health Sciences CenterShreveport, Louisiana

David L. McWhorter, PhDPhiladelphia College of Osteopathic MedicineGeorgia CampusSuwanee, Georgia

Joseph J. Maleszewski, MDCollege of Medicine, Mayo ClinicRochester, Minnesota

Fabiola Medeiros, MDCollege of Medicine, Mayo ClinicRochester, Minnesota

William D. Meek, PhDOklahoma State University, College of Osteopathic MedicineTulsa, Oklahoma

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Karuna Munjal, MDBaylor College of MedicineHouston, Texas

Lily J. Ning, MDUniversity of Medicine & Dentistry of New Jersey, New JerseyMedical SchoolNewark, New Jersey

Diego F. Nino, PhDLouisiana State University Health Sciences Center, DelgadoCommunity CollegeNew Orleans, Louisiana

Sasha N. Noe, DO, PhDSaint Leo UniversitySaint Leo, Florida

Joanne Orth, PhDTemple University School of MedicineDowningtown, Pennsylvania

Nalini Pather, PhDUniversity of New South WalesSidney, Australia

Tom P. Phillips, PhDUniversity of MissouriColumbia, Missouri

Stephen R. Planck, PhDOregon Health and Science UniversityPortland, Oregon

Dennifield W. Player, BSUniversity of FloridaGainesville, Florida

Harry H. Plymale, PhDSan Diego State UniversitySan Diego, California

Rebecca L. Pratt, PhDWest Virginia School of Osteopathic MedicineLewisburg, West Virginia

Margaret Pratten, PhDThe University of Nottingham, Medical SchoolNottingham, United Kingdom

Rongsun Pu, PhDKean UniversityEast Brunswick, New Jersey

Romano Regazzi, PhDUniversity of Lausanne, Faculty of Biology and MedicineLausanne, Switzerland

Mary Rheuben, PhDMichigan State UniversityEast Lansing, Michigan

Jeffrey L. Salisbury, PhDCollege of Medicine, Mayo ClinicRochester, Minnesota

Young-Jin Son, PhDDrexel UniversityPhiladelphia, Pennsylvania

David K. Saunders, PhDUniversity of Northern IowaCedar Falls, Iowa

John T. Soley, DVM, PhDUniversity of PretoriaPretoria, South Africa

Anca M. Stefan, MDTouro University College of MedicineHackensack, New Jersey

Alvin Telser, PhDNorthwestern University Medical SchoolChicago, Illinois

Barry Timms, PhDSanford School of Medicine, University of South DakotaVermillion, South Dakota

James J. Tomasek, PhDUniversity of Oklahoma Health Science CenterOklahoma City, Oklahoma

John Matthew Velkey, PhDUniversity of MichiganAnn Arbor, Michigan

Daniel W. Visscher, MDUniversity of Michigan Medical SchoolAnn Arbor, Michigan

Anne-Marie Williams, PhDUniversity of Tasmania, School of Medical SciencesHobart, Tasmania

Joan W. Witkin, PhDColumbia University, College of Physicians and SurgeonsNew York, New York

Alexandra P. Wolanskyj, MDCollege of Medicine, Mayo ClinicRochester, Minnesota

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Robert W. Zajdel, PhDState University of New York Upstate Medical UniversitySyracuse, New York

Renzo A. Zaldivar, MDAesthetic Facial & Ocular Plastic Surgery Center Chapel Hill, North Carolina

A few colleagues have made especially notable contributionsto this textbook. We are extremely grateful to Dr. Renzo Zal-divar from the Aesthetic Facial & Ocular Plastic Surgery Cen-ter in Chapel Hill, North Carolina for providing us withclinical images and content for several clinical correlations fold-ers in the chapter on the eye. Our deep appreciation goes toDrs. Fabiola Medeiros from Mayo Clinic and Donald Lowrie,Jr., from the University of Cincinnati College of Medicine forproviding original glass slides of the highest quality of severalspecimens. In addition, Todd Barnash from the Universityof Florida provided invaluable technical assistance with the

digitized text, figures, and photomicrographs. Thanks also goto Denny Player for his superb technical assistance with electron microscopy.

All of the new art in this edition was created by Rob Duck-wall and his wife Caitlin Duckwall from the Dragonfly MediaGroup (Baltimore, MD). Their expertise in creating innova-tive and aesthetically-pleasing artwork is greatly appreciated.

The authors also wish to extend special thanks to JenniferVerbiar, our managing editor, and her predecessor KathleenScogna, who provided expertise during the majority of the de-velopment process. Our editors problem solving and techni-cal skills were crucial to bringing this text to fruition, and theircontributions to the sixth edition were priceless. Our thanksgoes to Arijit Biswas, the Project Manager of MPS Limited,A Macmillan Company in New Delhi, India, and his staff ofcompositors for an excellent job in putting together this complex and challenging publication. Finally, a special thanksto Crystal Taylor for her support throughout the developmentof the book. Her diligence is much appreciated.

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6. CONNECTIVE TISSUE | 158

General Structure and Function of Connective Tissue | 158Embryonic Connective Tissue | 159Connective Tissue Proper | 160Connective Tissue Fibers | 161Extracellular Matrix | 173Connective Tissue Cells | 178Folder 6.1 Clinical Correlation: Collagenopathies | 170

Folder 6.2 Clinical Correlation: Sun Exposure and Molecular Changes in Photoaged Skin | 173

Folder 6.3 Clinical Correlation: Role of Myofibroblasts inWound Repair | 183

Folder 6.4 Functional Considerations: The MononuclearPhagocytotic System | 185

Folder 6.5 Clinical Correlation: The Role of Mast Cells and Basophils in Allergic Reactions | 188

Atlas PlatesPlate 4 Loose and Dense Irregular Connective

Tissue | 192

Plate 5 Dense Regular Connective Tissue, Tendons, andLigaments | 194

Plate 6 Elastic Fibers and Elastic Lamellae | 196

7. CARTILAGE | 198

Overview of Cartilage | 198Hyaline Cartilage | 199Elastic Cartilage | 204Fibrocartilage | 204Chondrogenesis and Cartilage Growth | 206Repair of Hyaline Cartilage | 207Folder 7.1 Clinical Correlation: Osteoarthritis | 199

Folder 7.2 Clinical Correlation: Malignant Tumors of the Cartilage; Chondrosarcomas | 208

Atlas PlatesPlate 7 Hyaline Cartilage | 210

Plate 8 Cartilage and the Developing Skeleton | 212

Plate 9 Elastic Cartilage | 214

Plate 10 Fibrocartilage | 216

8. BONE | 218

Overview of Bone | 218Bones and Bone Tissue | 219General Structure of Bones | 220Cells of Bone Tissue | 223Bone Formation | 232Biologic Mineralization and Matrix Vesicles | 241Physiologic Aspects of Bone | 242Folder 8.1 Clinical Correlation: Joint Diseases | 221

Folder 8.2 Clinical Correlation: Osteoporosis | 233

Folder 8.3 Clinical Correlation: Nutritional Factors in Bone Formation | 234

Folder 8.4 Functional Considerations: Hormonal Regulation of Bone Growth | 242

Atlas PlatesPlate 11 Bone, Ground Section | 244

Plate 12 Bone and Bone Tissue | 246

Plate 13 Endochondral Bone Formation I | 248

Plate 14 Endochondral Bone Formation II | 250

Plate 15 Intramembranous Bone Formation | 252

9. ADIPOSE TISSUE | 254

Overview of Adipose Tissue | 254White Adipose Tissue | 254Brown Adipose Tissue | 259Folder 9.1 Clinical Correlation: Obesity | 261

Folder 9.2 Clinical Correlation: Adipose Tissue Tumors | 262

Folder 9.3 Clinical Correlation: PET Scanning and Brown Adipose Tissue Interference | 264

Atlas PlatesPlate 16 Adipose Tissue | 266

10. BLOOD | 268

Overview of Blood | 268Plasma | 269Erythrocytes | 270Leukocytes | 275Thrombocytes | 286Formation of Blood Cells (Hemopoiesis) | 289Bone Marrow | 298Folder 10.1 Clinical Correlation: ABO and Rh Blood

Group Systems | 273

Folder 10.2 Clinical Correlation: Hemoglobin in Patients with Diabetes | 274

Folder 10.3 Clinical Correlation: Hemoglobin Disorders | 276

Folder 10.4 Clinical Correlation: Inherited Disorders ofNeutrophils; Chronic Granulomatous Disease(CGD) | 281

Folder 10.5 Clinical Correlation: Hemoglobin Breakdown and Jaundice | 281

Folder 10.6 Clinical Correlation: Cellularity of the Bone Marrow | 300

Atlas PlatesPlate 17 Erythrocytes and Granulocytes | 302

Plate 18 Agranulocytes and Red Marrow | 304

Plate 19 Erythropoiesis | 306

Plate 20 Granulopoiesis | 308

11. MUSCLE TISSUE | 310

Overview and Classification of Muscle | 310Skeletal Muscle | 311Cardiac Muscle | 327Smooth Muscle | 331Folder 11.1 Functional Considerations: Muscle Metabolism

and Ischemia | 316

Folder 11.2 Clinical Correlation: Muscular DystrophiesDystrophin and Dystrophin- Associated Proteins | 319

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1chapter 1Methods

OVERVIEW OF METHODS USEDIN HISTOLOGY / 1TISSUE PREPARATION / 2

Hematoxylin and Eosin Staining With FormalinFixation / 2

Other Fixatives / 2Other Staining Procedures / 3

HISTOCHEMISTRY AND CYTOCHEMISTRY / 3

Chemical Composition of Histologic Samples / 3Chemical Basis of Staining / 5Enzyme Digestion / 7Enzyme Histochemistry / 7Immunocytochemistry / 7Hybridization Techniques / 10Autoradiography / 12

MICROSCOPY / 13Light Microscopy / 13Examination of a Histologic Slide Preparation

in the Light Microscope / 14Other Optical Systems / 15Electron Microscopy / 18Atomic Force Microscopy / 20Folder 1.1 Clinical Correlation:

Frozen Sections / 4Folder 1.2 Functional Considerations:

Feulgen Microspectrophotometry / 7Folder 1.3 Clinical Correlation:

Monoclonal Antibodies in Medicine / 9Folder 1.4 Proper Use of the Light

Microscope / 11

many auxiliary techniques of cell and molecular biology.These auxiliary techniques include:

histochemistry and cytochemistry, immunocytochemistry and hybridization techniques, autoradiography, organ and tissue culture, cell and organelle separation by differential centrifugation,

and

specialized microscopic techniques and microscopes.The student may feel removed from such techniques and experimental procedures because direct experience with themis usually not available in current curricula. Nevertheless, it isimportant to know something about specialized proceduresand the data they yield. This chapter provides a survey of meth-ods and offers an explanation of how the data provided by thesemethods can help the student acquire a better understanding ofcells, tissues, and organ function.

One problem students in histology face is understandingthe nature of the two-dimensional image of a histologic slide

OVERVIEW OF METHODS USEDIN HISTOLOGY

The objective of a histology course is to lead the studentto understand the microanatomy of cells, tissues, and organs and to correlate structure with function.

The methods used by histologists are extremely diverse.Much of the histology course content can be framed in termsof light microscopy. Today, students in histology laboratoriesuse either light microscopes or, with increasing frequency,virtual microscopy, which represents a method of viewing adigitized microscopic specimen on a computer screen. In thepast, more detailed interpretation of microanatomy was withthe electron microscope (EM)both the transmissionelectron microscope (TEM) and the scanning electronmicroscope (SEM). Now the atomic force microscope(AFM) can also provide high-resolution images, which arecomparable in resolution to those obtained from TEM. BothEM and AFM, because of their greater resolution and usefulmagnification, are often the last step in data acquisition from

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or an electron micrograph and how the image relates to thethree-dimensional structure from which it came. To bridgethis conceptual gap, we must first present a brief descriptionof the methods by which slides and electron microscopicspecimens are produced.

TISSUE PREPARATION

Hematoxylin and Eosin Staining With Formalin Fixation

The routinely prepared hematoxylin and eosinstainedsection is the specimen most commonly studied.

The slide set given each student to study with the light micro-scope consists mostly of formalin-fixed, paraffin-embedded,hematoxylin and eosin (H&E)stained specimens. Nearly allof the light micrographs in the Atlas section of this book are ofslides from actual student sets. Also, most photomicrographsused to illustrate tissues and organs in histology lectures andconferences are taken from such slides. Other staining tech-niques are sometimes used to demonstrate specific cell and tis-sue components; several of these methods are discussed below.

The first step in preparation of a tissue or organ sample isfixation to preserve structure.

Fixation, usually by a chemical or mixture of chemicals, per-manently preserves the tissue structure for subsequent treat-ments. Specimens should be immersed in fixative immediatelyafter they are removed from the body. Fixation is used to:

terminate cell metabolism, prevent enzymatic degradation of cells and tissues by

autolysis (self-digestion),

kill pathogenic microorganisms such as bacteria, fungi,and viruses, and

harden the tissue as a result of either cross-linking or dena-turing protein molecules.

Formalin, a 37% aqueous solution of formaldehyde, at variousdilutions and in combination with other chemicals and buffers,is the most commonly used fixative. Formaldehyde preservesthe general structure of the cell and extracellular componentsby reacting with the amino groups of proteins (most oftencross-linked lysine residues). Because formaldehyde does notsignificantly alter their three-dimensional structure, proteinsmaintain their ability to react with specific antibodies. Thisproperty is important in immunocytochemical staining meth-ods (see page 7). The standard commercial solution offormaldehyde buffered with phosphates (pH 7) acts relativelyslowly but penetrates the tissue well. However, because it doesnot react with lipids, it is a poor fixative of cell membranes.

In the second step, the specimen is prepared for embed-ding in paraffin to permit sectioning.

Preparing a specimen for examination requires its infiltrationwith an embedding medium that allows it to be thinlysliced, typically in the range of 5 to 15 m (1 micrometer[m] equals 1/1,000 of a millimeter [mm]; see Table 1.1).The specimen is washed after fixation and dehydrated in a

series of alcohol solutions of ascending concentration as highas 100% alcohol to remove water. In the next step, clearing,organic solvents such as xylol or toluol, which are miscible inboth alcohol and paraffin, are used to remove the alcohol be-fore infiltration of the specimen with melted paraffin.

When the melted paraffin is cool and hardened, it istrimmed into an appropriately sized block. The block isthen mounted in a specially designed slicing machineamicrotomeand cut with a steel knife. The resulting sec-tions are then mounted on glass slides using mountingmedium (pinene or acrylic resins) as an adhesive.

In the third step, the specimen is stained to permit exam-ination.

Because paraffin sections are colorless, the specimen is not yetsuitable for light microscopic examination. To color or stain thetissue sections, the paraffin must be dissolved out, again withxylol or toluol, and the slide must then be rehydrated through aseries of solutions of descending alcohol concentration. The tis-sue on the slides is then stained with hematoxylin in water. Be-cause the counterstain, eosin, is more soluble in alcohol than inwater, the specimen is again dehydrated through a series of alco-hol solutions of ascending concentration and stained with eosinin alcohol. Figure 1.1 shows the results of staining with hema-toxylin alone, eosin alone, and hematoxylin with counterstaineosin. After staining, the specimen is then passed through xylolor toluol to a nonaqueous mounting medium and covered witha coverslip to obtain a permanent preparation.

Other Fixatives

Formalin does not preserve all cell and tissue components.

Although H&Estained sections of formalin-fixed specimensare convenient to use because they adequately display generalstructural features, they cannot elucidate the specific chemicalcomposition of cell components. Also, many components arelost in the preparation of the specimen. To retain these compo-nents and structures, other fixation methods must be used.These methods are generally based on a clear understanding ofthe chemistry involved. For instance, the use of alcohols andorganic solvents in routine preparations removes neutral lipids.

To retain neutral lipids, such as those in adipose cells, frozensections of formalin-fixed tissue and dyes that dissolve in fatsmust be used; to retain membrane structures, special fixatives

2

TABLE Commonly Used Linear Equivalents1.11 picometer (pm) 0.01 angstrom ()

1 angstrom 0.1 nanometer (nm)

10 angstroms 1.0 nanometer

1 nanometer 1,000 picometers

1,000 nanometers 1.0 micrometer (m)

1,000 micrometers 1.0 millimeter (mm)

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containing heavy metals that bind to the phospholipids, suchas permanganate and osmium, are used (Folder 1.1). The rou-tine use of osmium tetroxide as a fixative for electron mi-croscopy is the primary reason for the excellent preservation ofmembranes in electron micrographs.

Other Staining Procedures

Hematoxylin and eosin are used in histology primarily todisplay structural features.

Despite the merits of H&E staining, the procedure does notadequately reveal certain structural components of histologicsections such as elastic material, reticular fibers, basementmembranes, and lipids. When it is desirable to display thesecomponents, other staining procedures, most of them selec-tive, can be used. These procedures include the use of orceinand resorcin-fuchsin for elastic material and silver impregna-tion for reticular fibers and basement membrane material. Al-though the chemical bases of many staining methods are notalways understood, they work. Knowing the components thata procedure reveals is more important than knowing preciselyhow the procedure works.

HISTOCHEMISTRY ANDCYTOCHEMISTRY

Specific chemical procedures can provide informationabout the function of cells and the extracellular compo-nents of tissues.

Histochemical and cytochemical procedures may be based onspecific binding of a dye, use of a fluorescent dyelabeled

antibody with a particular cell component, or the inherentenzymatic activity of a cell component. In addition, manylarge molecules found in cells can be localized by the processof autoradiography, in which radioactively tagged precur-sors of the molecule are incorporated by cells and tissues be-fore fixation. Many of these procedures can be used with bothlight microscopic and electron microscopic preparations.

Before discussing the chemistry of routine staining andhistochemical and cytochemical methods, it is useful to ex-amine briefly the nature of a routinely fixed and embeddedsection of a specimen.

Chemical Composition of HistologicSamples

The chemical composition of a tissue ready for routinestaining differs from living tissue.

The components that remain after fixation consist mostly oflarge molecules that do not readily dissolve, especially aftertreatment with the fixative. These large molecules, particu-larly those that react with other large molecules to formmacromolecular complexes, are usually preserved in a tissuesection. Examples of such large macromolecular complexesinclude:

nucleoproteins formed from nucleic acids bound to protein,

intracellular cytoskeletal proteins complexed with as-sociated proteins,

extracellular proteins in large insoluble aggregates,bound to similar molecules by cross-linking of neighbor-ing molecules, as in collagen fiber formation, and

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FIGURE 1.1 Hematoxylin and eosin (H&E) staining. This series of specimens from the pancreas are serial (adjacent) sections thatdemonstrate the effect of hematoxylin and eosin used alone and hematoxylin and eosin used in combination. a. This photomicrographreveals the staining with hematoxylin only. Although there is a general overall staining of the specimen, those components and structuresthat have a high affinity for the dye are most heavily stainedfor example, the nuclear DNA and areas of the cell containing cytoplasmicRNA. b. In this photomicrograph, eosin, the counterstain, likewise has an overall staining effect when used alone. Note, however, thatthe nuclei are less conspicuous than in the specimen stained with hematoxylin alone. After the specimen is stained with hematoxylin andthen prepared for staining with eosin in alcohol solution, the hematoxylin that is not tightly bound is lost, and the eosin then stains thosecomponents to which it has a high affinity. c. This photomicrograph reveals the combined staining effect of H&E. 480.

a b ca b c

3

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4 membrane phospholipidprotein (or carbohydrate)complexes.

These molecules constitute the structure of cells and tissuesthat is, they make up the formed elements of the tissue. Theyare the basis for the organization that is seen in tissue with themicroscope.

In many cases, a structural element is also a functionalunit. For example, in the case of proteins that make up thecontractile filaments of muscle cells, the filaments are the vis-ible structural components and the actual participants in thecontractile process. The RNA of the cytoplasm is visualized as

part of a structural component (e.g., ergastoplasm of secre-tory cells, Nissl bodies of nerve cells) and is also the actualparticipant in the synthesis of protein.

Many tissue components are lost during the routinepreparation of H&Estained sections.

Despite the fact that nucleic acids, proteins, and phospho-lipids are mostly retained in tissue sections, many are alsolost. Small proteins and small nucleic acids, such as transferRNA, are generally lost during the preparation of the tissue.As previously described, neutral lipids are usually dissolved bythe organic solvents used in tissue preparation. Other large

Sometimes, the pathologist may be asked to immediatelyevaluate tissue obtained during surgery, especially when in-stant pathologic diagnosis may determine how the surgerywill proceed. There are several indications to perform suchan evaluation, routinely known as a frozen section. Mostcommonly, a surgeon in the operating room requests afrozen section when no preoperative diagnosis was availableor when unexpected intraoperative findings must be identi-fied. In addition, the surgeon may want to know whether all ofa pathologic mass within the healthy tissue limit has been re-moved and whether the margin of the surgical resection isfree of diseased tissue. Frozen sections are also done incombination with other procedures such as endoscopy orthin-needle biopsy to confirm whether the obtained biopsymaterial will be usable in further pathologic examinations.

Three main steps are involved in frozen section prepa-ration:

Freezing the tissue sample. Small tissue samples arefrozen either by using compressed carbon dioxide or byimmersion in a cold fluid (isopentane) at a temperature of

50C. Freezing can be achieved in a special high-efficiency refrigerator. Freezing makes the tissue solidand allows sectioning with a microtome.

Sectioning the frozen tissue. Sectioning is usually per-formed inside a cryostat, a refrigerated compartmentcontaining a microtome. Because the tissue is frozensolid, it can be cut into extremely thin (5 to 10 m) sec-tions. The sections are then mounted on glass slides.

Staining the cut sections. Staining is done to differen-tiate cell nuclei from the rest of the tissue. The mostcommon stains used for frozen sections are H&E,methylene blue (Fig. F1.1.1), and PAS stains.

The entire process of preparation and evaluation of frozensections may take as little as 10 minutes to complete. Thetotal time to obtain results largely depends on the transporttime of the tissue from the operating room to the pathologylaboratory, on the pathologic technique used, and the expe-rience of the pathologist. The findings are then directly com-municated to the surgeon waiting in the operating room.

FOLDER 1.1 Clinical Correlation: Frozen Sections

a ba b

FIGURE F1.1.1 Evaluation of aspecimen obtained during surgeryby frozen-section technique. a. Thisphotomicrograph shows a specimenobtained from the large intestine thatwas prepared by frozen-sectiontechnique and stained with methyleneblue. 160 b. Part of the specimenwas fixed in formalin and processed asa routine H&E preparation. Examinationof the frozen section revealed it to benormal. This diagnosis was laterconfirmed by examining the routinelyprepared H&E specimen. 180.(Courtesy of Dr. Daniel W. Visscher.)

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molecules also may be lost, for example, by being hydrolyzedbecause of the unfavorable pH of the fixative solutions. Ex-amples of large molecules lost during routine fixation inaqueous fixatives are:

glycogen (an intracellular storage carbohydrate commonin liver and muscle cells), and

proteoglycans and glycosaminoglycans (extracellularcomplex carbohydrates found in connective tissue).

These molecules can be preserved, however, by using a non-aqueous fixative for glycogen or by adding specific bindingagents to the fixative solution that preserve extracellular carbohydrate-containing molecules.

Soluble components, ions, and small molecules are alsolost during the preparation of paraffin sections.

Intermediary metabolites, glucose, sodium, chloride, andsimilar substances are lost during preparation of routineH&E paraffin sections. Many of these substances canbe studied in special preparations, sometimes with consider-able loss of structural integrity. These small soluble ions andmolecules do not make up the formed elements of a tissue;they participate in synthetic processes or cellular reactions.When they can be preserved and demonstrated by specificmethods, they provide invaluable information about cellmetabolism, active transport, and other vital cellular pro-cesses. Water, a highly versatile molecule, participates in thesereactions and processes and contributes to the stabilization ofmacromolecular structure through hydrogen bonding.

Chemical Basis of Staining

Acidic and Basic DyesHematoxylin and eosin are the most commonly used dyesin histology.

An acidic dye, such as eosin, carries a net negative charge onits colored portion and is described by the general formula[Nadye].

A basic dye carries a net positive charge on its colored por-tion and is described by the general formula [dyeCl].

Hematoxylin does not meet the definition of a strict basicdye but has properties that closely resemble those of a basicdye. The color of a dye is not related to whether it is basic oracidic, as can be noted by the examples of basic and acidicdyes listed in Table 1.2.

Basic dyes react with anionic components of cells and tissue (components that carry a net negative charge).

Anionic components include the phosphate groups of nu-cleic acids, the sulfate groups of glycosaminoglycans, and thecarboxyl groups of proteins. The ability of such anionic groupsto react with a basic dye is called basophilia [Gr., base-loving].Tissue components that stain with hematoxylin also exhibitbasophilia.

The reaction of the anionic groups varies with pH. Thus:

At a high pH (about 10), all three groups are ionized and avail-able for reaction by electrostatic linkages with the basic dye.

At a slightly acidic to neutral pH (5 to 7), sulfate and phos-phate groups are ionized and available for reaction with thebasic dye by electrostatic linkages.

At low pH (below 4), only sulfate groups remain ionizedand react with basic dyes.

Therefore, staining with basic dyes at a specific pH can be usedto focus on specific anionic groups; because the specific anionicgroups are found predominantly on certain macromolecules,the staining serves as an indicator of these macromolecules.

As mentioned, hematoxylin is not, strictly speaking, abasic dye. It is used with a mordant (i.e., an intermediatelink between the tissue component and the dye). The mor-dant causes the stain to resemble a basic dye. The linkage inthe tissuemordanthematoxylin complex is not a sim-ple electrostatic linkage; when sections are placed in water,hematoxylin does not dissociate from the tissue. Hema-toxylin lends itself to those staining sequences in which it isfollowed by aqueous solutions of acidic dyes. True basicdyes, as distinguished from hematoxylin, are not generallyused in sequences in which the basic dye is followed by anacidic dye. The basic dye then tends to dissociate from thetissue during the aqueous solution washes between the twodye solutions.

Acidic dyes react with cationic groups in cells and tissues,particularly with the ionized amino groups of proteins.

The reaction of cationic groups with an acidic dye is calledacidophilia [Gr., acid-loving]. Reactions of cell and tissuecomponents with acidic dyes are neither as specific nor as pre-cise as reactions with basic dyes.

Although electrostatic linkage is the major factor in the pri-mary binding of an acidic dye to the tissue, it is not the onlyone; because of this, acidic dyes are sometimes used in combi-nations to color different tissue constituents selectively. For ex-ample, three acidic dyes are used in the Mallory staining

TABLE Some Basic and AcidicDyes1.2Dye Color

Basic dyes

Methyl green Green

Methylene blue Blue

Pyronin G Red

Toluidine blue Blue

Acidic dyes

Acid fuchsin Red

Aniline blue Blue

Eosin Red

Orange G Orange

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6technique: aniline blue, acid fuchsin, and orange G. Thesedyes selectively stain collagen, ordinary cytoplasm, and redblood cells, respectively. Acid fuchsin also stains nuclei.

In other multiple acidic dye techniques, hematoxylin isused to stain nuclei first, and then acidic dyes are used to staincytoplasm and extracellular fibers selectively. The selectivestaining of tissue components by acidic dyes is attributable torelative factors such as the size and degree of aggregation ofthe dye molecules and the permeability and compactness ofthe tissue.

Basic dyes can also be used in combination or sequentially(e.g., methyl green and pyronin to study protein synthesisand secretion), but these combinations are not as widely usedas acidic dye combinations.

A limited number of substances within cells and the extra-cellular matrix display basophilia.

These substances include:

heterochromatin and nucleoli of the nucleus (chiefly because of ionized phosphate groups in nucleic acids ofboth),

cytoplasmic components such as the ergastoplasm (alsobecause of ionized phosphate groups in ribosomal RNA),and

extracellular materials such as the complex carbohy-drates of the matrix of cartilage (because of ionized sulfategroups).

Staining with acidic dyes is less specific, but more sub-stances within cells and the extracellular matrix exhibitacidophilia.

These substances include:

most cytoplasmic filaments, especially those of musclecells,

most intracellular membranous components andmuch of the otherwise unspecialized cytoplasm, and

most extracellular fibers (primarily because of ionizedamino groups).

MetachromasiaCertain basic dyes react with tissue components that shifttheir normal color from blue to red or purple; this ab-sorbance change is called metachromasia.

The underlying mechanism for metachromasia is the pres-ence of polyanions within the tissue. When these tissues arestained with a concentrated basic dye solution, such as tolui-dine blue, the dye molecules are close enough to formdimeric and polymeric aggregates. The absorption propertiesof these aggregations differ from those of the individualnonaggregated dye molecules.

Cell and tissue structures that have high concentrationsof ionized sulfate and phosphate groupssuch as theground substance of cartilage, heparin-containing granulesof mast cells, and rough endoplasmic reticulum of plasmacellsexhibit metachromasia. Therefore, toluidine bluewill appear purple to red when it stains these components.

Aldehyde Groups and the Schiff ReagentThe ability of bleached basic fuchsin (Schiff reagent) toreact with aldehyde groups results in a distinctive redcolor and is the basis of the periodic acidSchiff and Feul-gen reactions.

The periodic acidSchiff (PAS) reaction stains carbohy-drates and carbohydrate-rich macromolecules. It is used todemonstrate glycogen in cells, mucus in various cells and tissues,the basement membrane that underlies epithelia, and reticularfibers in connective tissue. The Feulgen reaction, which relieson a mild hydrochloric acid hydrolysis, is used to stain DNA.

The PAS reaction is based on the following facts:

Hexose rings of carbohydrates contain adjacent carbons,each of which bears a hydroxyl (OH) group.

Hexosamines of glycosaminoglycans contain adjacent car-bons, one of which bears an OH group, whereas the otherbears an amino (NH2) group.

Periodic acid cleaves the bond between these adjacent car-bon atoms and forms aldehyde groups.

These aldehyde groups react with the Schiff reagent to givea distinctive magenta color.

The PAS staining of basement membrane (Fig. 1.2) and retic-ular fibers is based on the content or association of proteogly-cans (complex carbohydrates associated with a protein core).PAS staining is an alternative to silver-impregnation meth-ods, which are also based on reaction with the sugarmolecules in the proteoglycans.

The Feulgen reaction is based on the cleavage of purinesfrom the deoxyribose of DNA by mild acid hydrolysis; thesugar ring then opens with the formation of aldehyde groups.Again, the newly formed aldehyde groups react with the

FIGURE 1.2 Photomicrograph of kidney tissue stained bythe PAS method. This histochemical method demonstrates andlocalizes carbohydrates and carbohydrate-rich macromolecules.The basement membranes are PAS positive as evidenced by themagenta staining of these sites. The kidney tubules (T ) are sharplydelineated by the stained basement membrane surrounding thetubules. The glomerular capillaries (C) and the epithelium ofBowmans capsule (BC) also show PAS-positive basementmembranes. 360.

T

T

T T

T

C

CBC

T

T

T T

T

C

CBC

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Schiff reagent to give the distinctive magenta color. The reac-tion of the Schiff reagent with DNA is stoichiometric,meaning that the product of this reaction is measurable andproportional to the amount of DNA. It can be used, there-fore, in spectrophotometric methods to quantify the amountof DNA in the nucleus of a cell. RNA does not stain with theSchiff reagent because it lacks deoxyribose.

Enzyme Digestion

Enzyme digestion of a section adjacent to one stained for aspecific componentsuch as glycogen, DNA, or RNAcan be used to confirm the identity of the stained material.

Intracellular material that stains with the PAS reaction maybe identified as glycogen by pretreatment of sections with di-astase or amylase. Abolition of the staining after these treat-ments positively identifies the stained material as glycogen.

Similarly, pretreatment of tissue sections with deoxyri-bonuclease (DNAse) will abolish the Feulgen staining inthose sections, and treatment of sections of protein secretoryepithelia with ribonuclease (RNAse) will abolish the stainingof the ergastoplasm with basic dyes.

Enzyme Histochemistry

Histochemical methods are also used to identify and localize enzymes in cells and tissues.

To localize enzymes in tissue sections, special care must betaken in fixation to preserve the enzyme activity. Usually,mild aldehyde fixation is the preferred method. In these pro-cedures, the reaction product of the enzyme activity, ratherthan the enzyme itself, is visualized. In general, a capturereagent, either a dye or a heavy metal, is used to trap or bindthe reaction product of the enzyme by precipitation at the site

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Feulgen microspectrophotometry is a technique devel-oped to study DNA increases in developing cells and toanalyze ploidythat is, the number of times the normal DNAcontent of a cell is multiplied (a normal, nondividing cell issaid to be diploid; a sperm or egg cell is haploid ). Twotechniques, static cytometry for tissue sections and flowcytometry for isolated cells, are used to quantify theamount of nuclear DNA. The technique of static cytometryof Feulgen-stained sections of tumors uses microspec-trophotometry coupled with a digitizing imaging system tomeasure the absorption of light emitted by cells and cellclusters at 560-nm wavelength. In contrast, the flow cy-tometry technique uses instrumentation able to scan onlysingle cells flowing past a sensor in a liquid medium. Thistechnique provides rapid, quantitative analysis of a singlecell based on the measurement of fluorescent light emis-

sion. Currently, Feulgen microspectrophotometry is usedto study changes in the DNA content in dividing cells un-dergoing differentiation. It is also used clinically to analyzeabnormal chromosomal number (i.e., ploidy patterns) inmalignant cells. Some malignant cells that have a largelydiploid pattern are said to be well differentiated; tumorswith these types of cells have a better prognosis than tu-mors with aneuploid (nonintegral multiples of the haploidamount of DNA) and tetraploid cells. Feulgen microspec-trophotometry has been particularly useful in studies ofspecific adenocarcinomas (epithelial cancers), breast cancer, kidney cancer, colon and other gastrointestinalcancers, endometrial (uterine epithelium) cancer, and ovar-ian cancer. It is one of the most valuable tools for patholo-gists in evaluating the metastatic potential of these tumorsand in making prognostic and treatment decisions.

FOLDER 1.2 Functional Considerations: FeulgenMicrospectrophotometry

of reaction. In a typical reaction to display a hydrolytic en-zyme, the tissue section is placed in a solution containing asubstrate (AB) and a trapping agent (T) that precipitates oneof the products as follows:

enzymeAB T AT B

where AT is the trapped end product and B is the hydrolyzedsubstrate.

By using such methods, the lysosome, first identified indifferential centrifugation studies of cells, was equated with avacuolar component seen in electron micrographs. In lightlyfixed tissues, the acid hydrolases and esterases contained inlysosomes react with an appropriate substrate. The reactionmixture also contains lead ions to precipitate (e.g., lead phos-phate derived from the action of acid phosphatase). The pre-cipitated reaction product can then be observed with bothlight and electron microscopy.

Similar light and electron microscopy histochemical pro-cedures have been developed to demonstrate alkaline phos-phatase, adenosine triphosphatases (ATPases) of manyvarieties (including the Na/K-ATPase that is the enzymaticbasis of the sodium pump in cells and tissues), various es-terases, and many respiratory enzymes (Fig. 1.3).

Immunocytochemistry

The specificity of a reaction between an antigen and an an-tibody is the underlying basis of immunocytochemistry.

Antibodies, also known as immunoglobulins, are glyco -proteins that are produced by specific cells of the immunesystem in response to a foreign protein, or antigen. In thelaboratory, antibodies can be purified from the blood andconjugated (attached) to a fluorescent dye. In general, fluo-rescent dyes (fluorochromes) are chemicals that absorb

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light of different wavelengths (e.g., ultraviolet light) and thenemit visible light of a specific wavelength (e.g., green, yellow,red). Fluorescein, the most commonly used dye, absorbs ul-traviolet light and emits green light. Antibodies conjugatedwith fluorescein can be applied to sections of lightly fixed orfrozen tissues on glass slides to localize an antigen in cells andtissues. The reaction of antibody with antigen can then be ex-amined and photographed with a fluorescence microscope or confocal microscope that produces a three-dimensional reconstruction of the examined tissue (Fig. 1.4).

Two types of antibodies are used in immunocytochemistry:polyclonal antibodies that are produced by immunized an-imals and monoclonal antibodies that are produced by im-mortalized (continuously replicating) antibody-producingcell lines.

In a typical procedure, a specific protein, such as actin, is iso-lated from a muscle cell of one species, such as a rat, and in-jected into the circulation of another species, such as a rabbit.In the immunized rabbit, the rats actin molecules are recog-nized by the rabbit immune system as a foreign antigen. Thisrecognition triggers a cascade of immunologic reactions in-volving multiple groups (clones) of immune cells called Blymphocytes. The cloning of B lymphocytes eventuallyleads to the production of anti-actin antibodies. Collectively,these polyclonal antibodies represent mixtures of differentantibodies produced by many clones of B lymphocytes that

each recognize different regions of the actin molecule. Theantibodies are then removed from the blood, purified, andconjugated with a fluorescent dye. They can now be used tolocate actin molecules in rat tissues or cells. If actin is presentin a cell or tissue, such as a fibroblast in connective tissue,then the fluorescein-labeled antibody binds to it and the reac-tion is visualized by fluorescence microscopy.

Monoclonal antibodies (Folder 1.3) are those producedby an antibody-producing cell line consisting of a singlegroup (clone) of identical B lymphocytes. The single clonethat becomes a cell line is obtained from an individual with multiple myeloma, a tumor derived from a single antibody-producing plasma cell. Individuals with multiple myelomasproduce a large population of identical, homogeneous anti-bodies with an identical specificity against an antigen. Toproduce monoclonal antibodies against a specific antigen, amouse or rat is immunized with that antigen. The activated Blymphocytes are then isolated from the lymphatic tissue(spleen or lymph nodes) of the animal and fused with themyeloma cell line. This fusion produces a hybridoma, an immortalized individual antibody-secreting cell line. To ob-tain monoclonal antibodies against rat actin molecules, forexample, the B lymphocytes from the lymphatic organs ofimmunized rabbits must be fused with myeloma cells.

8

FIGURE 1.3 Electron histochemical procedure for localizationof membrane ATPase in epithelial cells of rabbit gallbladder.Dark areas visible on the electron micrograph show the locationof the enzyme ATPase. This enzyme is detected in the plasmamembrane at the lateral domains of epithelial cells, whichcorrespond to the location of sodium pumps. These epithelialcells are involved in active transport of molecules across theplasma membrane. 26,000.

FIGURE 1.4 Confocal microscopy image of a rat cardiacmuscle cell. This image was obtained from the confocalmicroscope using the indirect immunofluorescence method. Twoprimary antibodies were used. The first primary antibody recognizesa specific lactate transporter (MCT1) and is detected with asecondary antibody conjugated with rhodamine (red). The secondprimary antibody is directed against the transmembrane proteinCD147, which is tightly associated with MCT1. This antibodywas detected by a secondary antibody labeled with fluorescein(green). The yellow color is visible at the point at which the twolabeled secondary antibodies exactly co-localize within the cardiacmuscle cell. This three-dimensional image shows that both proteinsare distributed on the surface of the muscle cell, whereas thelactate transporter alone is visible deep to the plasma membrane.(Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.)

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Both direct and indirect immunocytochemical methodsare used to locate a target antigen in cells and tissues.

The oldest immunocytochemistry technique used for identi-fying the distribution of an antigen within cells and tissues isknown as direct immunofluorescence. This technique usesa fluorochrome-labeled primary antibody (either polyclonalor monoclonal) that reacts with the antigen within the sample(Fig. 1.5a). As a one-step procedure, this method involves onlya single labeled antibody. Visualization of structures is notideal because of the low intensity of the signal emission. Directimmunofluorescence methods are now being replaced by theindirect method because of suboptimal sensitivity.

Indirect immunofluorescence provides much greatersensitivity than direct methods and is often referred to as thesandwich or double-layer technique. Instead of conjugatinga fluorochrome with a specific (primary) antibody directedagainst the antigen of interest (e.g., a rat actin molecule), the

fluorochrome is conjugated with a secondary antibody di-rected against rat primary antibody (i.e., goat anti-rat antibody;Fig. 1.5b). Therefore, when the fluorescein is conjugated di-rectly with the specific primary antibody, the method is direct;when fluorescein is conjugated with a secondary antibody, themethod is indirect. The indirect method considerably enhancesthe fluorescence signal emission from the tissue. An additionaladvantage of the indirect labeling method is that a single sec-ondary antibody can be used to localize the tissue-specificbinding of several different primary antibodies (Fig. 1.6). Formicroscopic studies, the secondary antibody can be conjugatedwith different fluorescent dyes so that multiple labels can beshown in the same tissue section (see Fig. 1.4). Drawbacks ofindirect immunofluorescence are that it is expensive, labor in-tensive, and not easily adapted to automated procedures.

It is also possible to conjugate polyclonal or mono-clonal antibodies with other substances, such as enzymes

FOLDER 1.3 Clinical Correlation: Monoclonal Antibodies in Medicine

Monoclonal antibodies are now widely used in im-munocytochemical techniques and also have many clini-cal applications. Monoclonal antibodies conjugated withradioactive compounds are used to detect and diagnosetumor metastasis in pathology, differentiate subtypes oftumors and stages of their differentiation, and in infec-

tious disease diagnosis to identify microorganisms inblood and tissue fluids. In recent clinical studies, mono-clonal antibodies conjugated with immunotoxins,chemotherapy agents, or radioisotopes have been usedto deliver therapeutic agents to specific tumor cells in thebody.

FIGURE 1.5 Direct and indirect immunofluorescence. a. In direct immunofluorescence, a fluorochrome-labeled primary antibodyreacts with a specific antigen within the tissue sample. Labeled structures are then observed in the fluorescence microscope in which anexcitation wavelength (usually ultraviolet light) triggers the emission of another wavelength. The length of this wavelength depends on thenature of the fluorochrome used for antibody labeling. b. The indirect method involves two processes. First, the specific primary antibodiesreact with the antigen of interest. Second, the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies.The visualization of labeled structures within the tissue is the same in both methods and requires the fluorescence microscope.

DIRECT IMMUNOFLUORESCENCE

Antigen

Antibody

Primaryantibody

Flourescent secondaryantibody

INDIRECT IMMUNOFLUORESCENCE

a

b

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(e.g., horseradish peroxidase), that convert colorless substratesinto an insoluble product of a specific color that precipitates atthe site of the enzymatic reaction. The staining that resultsfrom this immunoperoxidase method can be observed inthe light microscope (Folder 1.4) with either direct or indirectimmunocytochemical methods. In another variation, colloidalgold or ferritin (an iron-containing molecule) can be attachedto the antibody molecule. These electron-dense markers canbe visualized directly with the electron microscope.

Hybridization Techniques

Hybridization is a method of localizing messenger RNA(mRNA) or DNA by hybridizing the sequence of interestto a complementary strand of a nucleotide probe.

In general, the term hybridization describes the ability ofsingle-stranded RNA or DNA molecules to interact (hy-bridize) with complementary sequences. In the laboratory,hybridization requires the isolation of DNA or RNA, whichis then mixed with a complementary nucleotide sequence(called a nucleotide probe). Hybrids are detected mostoften using a radioactive label attached to one component ofthe hybrid.

Binding of the probe and sequence can take place in a solution or on a nitrocellulose membrane. In in situ

hybridization, the binding of the nucleotide probe to theDNA or RNA sequence of interest is performed within cellsor tissues, such as cultured cells or whole embryos. This tech-nique allows the localization of specific nucleotide sequencesas small as 10 to 20 copies of mRNA or DNA per cell.

Several nucleotide probes are used in in situ hybridiza-tion. Oligonucleotide probes can be as small as 20 to40 base pairs. Single- or double-stranded DNA probesare much longer and can contain as many as 1,000 basepairs. For specific localization of mRNA, complementaryRNA probes are used. These probes are labeled with ra-dioactive isotopes (e.g., 32P, 35S, 3H), a specifically modifiednucleotide (digoxigenin), or biotin (a commonly used cova-lent multipurpose label). Radioactive probes can be detectedand visualized by autoradiography. Digoxigenin and biotinare detected by immunocytochemical and cytochemicalmethods, respectively.

The strength of the bonds between the probe and thecomplementary sequence depends on the type of nucleic acidin the two strands. The strongest bond is formed between aDNA probe and a complementary DNA strand and theweakest between an RNA probe and a complementary RNAstrand. If a tissue specimen is expected to contain aminute amount of mRNA or a viral transcript, then poly-merase chain reaction (PCR) amplification for DNA orreverse transcriptase-PCR (RT-PCR) for RNA can beused. The amplified transcripts obtained during these proce-dures are usually detected using labeled complementary nu-cleotide probes in standard in situ hybridization techniques.

Recently, fluorescent dyes have been combined with nu-cleotide probes, making it possible to visualize multipleprobes at the same time (Fig. 1.7). This technique, called

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FIGURE 1.6 Microtubules visualized by immunocytochemicalmethods. The behavior of microtubules (elements of the cellcytoskeleton) obtained from human breast tumor cells can bestudied in vitro by measuring their nucleation activity, which isinitiated by the centrosome. This image was photographed in thefluorescence microscope. By use of indirect immunofluorescencetechniques, microtubules were labeled with a mixture of anti-tubulin and anti-tubulin monoclonal antibodies (primaryantibodies) and visualized by secondary antibodies conjugatedwith fluorescein dye (fluorescein isothiocyanategoat anti-mouseimmunoglobulin G). The antigenantibody reaction, performeddirectly on the glass coverslip, results in visualization of tubulinmolecules responsible for the formation of more than 120microtubules visible on this image. They originate from thecentriole and extend outward approximately 20 to 25 m in auniform radial array. 1,400. (Photomicrograph courtesy ofDrs. Wilma L. Lingle and Vivian A. Negron.)

FIGURE 1.7 Example of the FISH technique used in aprenatal screening test. Interphase nuclei of cells obtained fromamniotic fluid specimens were hybridized with two specific DNAprobes. The orange probe (LSI 21) is locus specific forchromosome 21, and the green probe (LSI 13) is locus specificfor chromosome 13. The right nucleus is from a normal amnioticfluid specimen and exhibits two green and two orange signals,which indicates two copies of chromosomes 13 and 21,respectively. The nucleus on the left has three orange signals,which indicate trisomy 21 (Down syndrome). DNA has beencounterstained with a nonspecific blue stain (DAPI stain) to makethe nucleus visible. 1,250. (Courtesy of Dr. Robert B. Jenkins.)

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FOLDER 1.4 Proper Use of the Light Microscope

continued next page

This brief introduction to the proper use of the light micro-scope is directed to those students who will use the micro-scope for the routine examination of tissues. If the followingcomments appear elementary, it is only because mostusers of the microscope fail to use it to its fullest advan-tage. Despite the availability of todays fine equipment, rel-atively little formal instruction is given on the correct use ofthe light microscope.

Expensive and highly corrected optics perform optimallyonly when the illumination and observation beam paths arecentered and properly adjusted. The use of proper settingsand proper alignment of the optic pathway will contributesubstantially to the recognition of minute details in thespecimen and to the faithful display of color for the visualimage and for photomicrography.

Khler illumination is one key to good microscopyand is incorporated in the design of practically all modernlaboratory and research microscopes. Figure F1.4.1 showsthe two light paths and all the controls for alignment on amodern laboratory microscope; it should be referred to infollowing the instructions given below to provide appropri-ate illumination in your microscope.

The alignment steps necessary to achieve good Khlerillumination are few and simple:

Focus the specimen. Close the field diaphragm. Focus the condenser by moving it up or down until the

outline of its field diaphragm appears in sharp focus.

Center the field diaphragm with the centering controls onthe (condenser) substage. Then open the field diaphragmuntil the light beam covers the full field observed.

Remove the eyepiece (or use a centering telescope or aphase telescope accessory if available) and observe theexit pupil of the objective. You will see an illuminated cir-cular field that has a radius directly proportional to thenumeric aperture of the objective. As you close the con-denser diaphragm, its outline will appear in this circularfield. For most stained materials, set the condenser di-aphragm to cover approximately two thirds of the objec-tive aperture. This setting results in the best compromisebetween resolution and contrast (contrast simply beingthe intensity difference between dark and light areas inthe specimen).

FIGURE F1.4.1 Diagram of a typical light microscope. This drawing shows a cross-sectional view of themicroscope, its operating components, and light path. (Courtesy of Carl Zeiss, Inc., Thornwood, NY.)

eyepiece

final image

exit pupil(eyepoint)

real interme-diate image

exit pupil of objective

specimen

condenserdiaphragm

fielddiaphragm

light source

focusingcontrol

lightsource

tube

objective

auxiliarycondenserlens

stagecondenserdiaphragm

condenserstage controlfield diaphragm

KHLER ILLUMINATION THROUGH THE MICROSCOPE IMAGINGBEAM PATHILLUMINATING

BEAM PATH

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FOLDER 1.4 Proper Use of the Light Microscope (Cont.)

Using only these five simple steps, the image obtained willbe as good as the optics allow. Now let us find out why.

First, why do we adjust the field diaphragm to cover onlythe field observed? Illuminating a larger field than the opticscan see only leads to internal reflections or stray light, re-sulting in more noise or a decrease in image contrast.

Second, why do we emphasize the setting of the con-denser diaphragmthat is, the illuminating aperture? Thisdiaphragm greatly influences the resolution and the con-trast with which specimen detail can be observed.

For most practical applications, the resolution is deter-mined by the equation

d NAobjective NAcondenser

whered point-to-point distance of resolved detail (in nm),

wavelength of light used (green 540 nm),

NA numeric aperture or sine of half angle picked upby the objective or condenser of a central speci-men point multiplied by the refractive index of themedium between objective or condenser andspecimen.

How do wavelength and numeric aperture directly influ-ence resolution? Specimen structures diffract light. Thediffraction angle is directly proportional to the wavelengthand inversely proportional to the spacing of the structures.According to physicist Ernst Abb, a given structural spac-ing can be resolved only when the observing optical sys-tem (objective) can see some of the diffracted lightproduced by the spacing. The larger the objectives aper-ture, the more diffracted the light that participates in theimage formation, resulting in resolution of smaller detailand sharper images.

Our simple formula, however, shows that the con-denser aperture is just as important as the objective aper-ture. This point is only logical when you consider thediffraction angle for an oblique beam or one of higheraperture. This angle remains essentially constant but ispresented to the objective in such a fashion that it can bepicked up easily.

How does the aperture setting affect the contrast? The-oretically, the best contrast transfer from object to imagewould be obtained by the interaction (interference) be-tween nondiffracted and all the diffracted wave fronts.

For the transfer of contrast between full transmissionand complete absorption in a specimen, the intensity rela-tionship between diffracted and nondiffracted light wouldhave to be 1:1 to achieve full destructive interference(black) or full constructive interference (bright). Whenthe condenser aperture matches the objective aperture,the nondiffracted light enters the objective with full inten-sity, but only part of the diffracted light can enter, resultingin decreased contrast. In other words, closing the apertureof the condenser to two thirds of the objective aperturebrings the intensity relationship between diffracted andnondiffracted light close

Documents

Ross Histology Text and Atlas 6th(1)