Junqueira's Basic Histology Text and Atlas, 14th Edition (PDF)

 CONTENTS i FOURTEENTH EDITION Junqueira’s Basic Histology T E X T A N D AT L A S Anthony L...

 CONTENTS i FOURTEENTH EDITION Junqueira’s Basic Histology T E X T A N D AT L A S Anthony L. Mescher, PhD Professor of Anatomy and Cell Biology Indiana University School of Medicine Bloomington, Indiana New York Chicago San Francisco Athens London Madrid Mexico City Milan New Delhi Singapore Sydney Toronto Copyright © 2016 by McGraw-Hill Education. All rights reserved. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publisher, with the exception that the program listings may be entered, stored, and executed in a computer system, but they may not be reproduced for publication. 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This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise. Contents KEY FEATURESâ ‡ VIâ ‡ |â ‡ PREFACEâ ‡IXâ ‡|â ‡ACKNOWLEDGMENTSâ ‡XI 1 Histology & Its Methods 5 Connective Tissueâ ‡ 96 of Studyâ ‡ 1 Cells of Connective Tissueâ ‡ 96 Preparation of Tissues for Studyâ ‡ 1 Fibersâ ‡103 Light Microscopyâ ‡ 4 Ground Substanceâ ‡ 111 Electron Microscopyâ ‡ 8 Types of Connective Tissueâ ‡ 114 Autoradiographyâ ‡9 Summary of Key Pointsâ ‡ 119 Cell & Tissue Cultureâ ‡ 10 Assess Your Knowledgeâ ‡ 120 Enzyme Histochemistryâ ‡ 10 Visualizing Specific Moleculesâ ‡ 10 6 Adipose Tissueâ ‡ 122 Interpretation of Structures in Tissue White Adipose Tissueâ ‡ 122 Sectionsâ ‡14 Brown Adipose Tissueâ ‡ 126 Summary of Key Pointsâ ‡ 15 Summary of Key Pointsâ ‡ 127 Assess Your Knowledgeâ ‡ 16 Assess Your Knowledgeâ ‡ 128 2 The Cytoplasmâ ‡ 17 7 Cartilageâ ‡129 Cell Differentiationâ ‡ 17 Hyaline Cartilageâ ‡ 129 The Plasma Membraneâ ‡ 17 Elastic Cartilageâ ‡ 133 Cytoplasmic Organellesâ ‡ 27 Fibrocartilageâ ‡134 The Cytoskeletonâ ‡ 42 Cartilage Formation, Growth, & Repairâ ‡ 134 Inclusionsâ ‡47 Summary of Key Pointsâ ‡ 136 Summary of Key Pointsâ ‡ 51 Assess Your Knowledgeâ ‡ 136 Assess Your Knowledgeâ ‡ 52 8 Boneâ ‡138 3 The Nucleusâ ‡ 53 Bone Cellsâ ‡ 138 Components of the Nucleusâ ‡ 53 Bone Matrixâ ‡ 143 The Cell Cycleâ ‡ 58 Periosteum & Endosteumâ ‡ 143 Mitosisâ ‡61 Types of Boneâ ‡ 143 Stem Cells & Tissue Renewalâ ‡ 65 Osteogenesisâ ‡148 Meiosisâ ‡65 Bone Remodeling & Repairâ ‡ 152 Apoptosisâ ‡67 Metabolic Role of Boneâ ‡ 153 Summary of Key Pointsâ ‡ 69 Jointsâ ‡155 Assess Your Knowledgeâ ‡ 70 Summary of Key Pointsâ ‡ 158 Assess Your Knowledgeâ ‡ 159 4 Epithelial Tissueâ ‡ 71 Characteristic Features of Epithelial Cellsâ ‡ 72 9 Nerve Tissue & the Nervous Specializations of the Apical Cell Surfaceâ ‡ 77 Systemâ ‡161 Types of Epitheliaâ ‡ 80 Development of Nerve Tissueâ ‡ 161 Transport Across Epitheliaâ ‡ 88 Neuronsâ ‡163 Renewal of Epithelial Cellsâ ‡ 88 Glial Cells & Neuronal Activityâ ‡ 168 Summary of Key Pointsâ ‡ 90 Central Nervous Systemâ ‡ 175 Assess Your Knowledgeâ ‡ 93 Peripheral Nervous Systemâ ‡ 182 iii iv CONTENTS Neural Plasticity & Regenerationâ ‡ 187 15 Digestive Tractâ ‡ 295 Summary of Key Pointsâ ‡ 190 General Structure of the Digestive Tractâ ‡ 295 Assess Your Knowledgeâ ‡ 191 Oral Cavityâ ‡ 298 Esophagusâ ‡305 10 Muscle Tissueâ ‡ 193 Stomachâ ‡307 Skeletal Muscleâ ‡ 193 Small Intestineâ ‡ 314 Cardiac Muscleâ ‡ 207 Large Intestineâ ‡ 318 Smooth Muscleâ ‡ 208 Summary of Key Pointsâ ‡ 326 Regeneration of Muscle Tissueâ ‡ 213 Assess Your Knowledgeâ ‡ 328 Summary of Key Pointsâ ‡ 213 Assess Your Knowledgeâ ‡ 214 16 Organs Associated with the Digestive Tractâ ‡329 11 The Circulatory Systemâ ‡ 215 Salivary Glandsâ ‡ 329 Heartâ ‡215 Pancreasâ ‡332 Tissues of the Vascular Wallâ ‡ 219 Liverâ ‡335 Vasculatureâ ‡220 Biliary Tract & Gallbladderâ ‡ 345 Lymphatic Vascular Systemâ ‡ 231 Summary of Key Pointsâ ‡ 346 Summary of Key Pointsâ ‡ 235 Assess Your Knowledgeâ ‡ 348 Assess Your Knowledgeâ ‡ 235 17 The Respiratory Systemâ ‡ 349 12 Bloodâ ‡ 237 Nasal Cavitiesâ ‡ 349 Composition of Plasmaâ ‡ 237 Pharynxâ ‡352 Blood Cellsâ ‡ 239 Larynxâ ‡352 Summary of Key Pointsâ ‡ 250 Tracheaâ ‡354 Assess Your Knowledgeâ ‡ 252 Bronchial Tree & Lungâ ‡ 354 Lung Vasculature & Nervesâ ‡ 366 13 Hemopoiesisâ ‡ 254 Pleural Membranesâ ‡ 368 Stem Cells, Growth Factors, & Differentiationâ ‡ 254 Respiratory Movementsâ ‡ 368 Bone Marrowâ ‡ 255 Summary of Key Pointsâ ‡ 369 Maturation of Erythrocytesâ ‡ 258 Assess Your Knowledgeâ ‡ 369 Maturation of Granulocytesâ ‡ 260 Maturation of Agranulocytesâ ‡ 263 18 Skinâ ‡ 371 Origin of Plateletsâ ‡ 263 Epidermisâ ‡372 Summary of Key Pointsâ ‡ 265 Dermisâ ‡378 Assess Your Knowledgeâ ‡ 265 Subcutaneous Tissueâ ‡ 381 Sensory Receptorsâ ‡ 381 14 The Immune System & Lymphoid Hairâ ‡383 Organsâ ‡267 Nailsâ ‡384 Innate & Adaptive Immunityâ ‡ 267 Skin Glandsâ ‡ 385 Cytokinesâ ‡269 Skin Repairâ ‡ 388 Antigens & Antibodiesâ ‡ 270 Summary of Key Pointsâ ‡ 391 Antigen Presentationâ ‡ 271 Assess Your Knowledgeâ ‡ 391 Cells of Adaptive Immunityâ ‡ 273 Thymusâ ‡276 19 The Urinary Systemâ ‡ 393 Mucosa-Associated Lymphoid Tissueâ ‡ 281 Kidneysâ ‡393 Lymph Nodesâ ‡ 282 Blood Circulationâ ‡ 394 Spleenâ ‡286 Renal Function: Filtration, Secretion, & Summary of Key Pointsâ ‡ 293 Reabsorptionâ ‡395 Assess Your Knowledgeâ ‡ 294 Ureters, Bladder, & Urethraâ ‡ 406  CONTENTS v Summary of Key Pointsâ ‡ 411 22 The Female Reproductive Systemâ ‡ 460 Assess Your Knowledgeâ ‡ 412 Ovariesâ ‡460 Uterine Tubesâ ‡ 470 20 Endocrine Glandsâ ‡ 413 Major Events of Fertilizationâ ‡ 471 Pituitary Gland (Hypophysis)â ‡ 413 Uterusâ ‡471 Adrenal Glandsâ ‡ 423 Embryonic Implantation, Decidua, & the Placentaâ ‡ 478 Pancreatic Isletsâ ‡ 427 Cervixâ ‡482 Diffuse Neuroendocrine Systemâ ‡ 429 Vaginaâ ‡483 Thyroid Glandâ ‡ 429 External Genitaliaâ ‡ 483 Parathyroid Glandsâ ‡ 432 Mammary Glandsâ ‡ 483 Pineal Glandâ ‡ 434 Summary of Key Pointsâ ‡ 488 Summary of Key Pointsâ ‡ 437 Assess Your Knowledgeâ ‡ 489 Assess Your Knowledgeâ ‡ 437 23 The Eye & Ear: Special Sense 21 The Male Reproductive Organsâ ‡490 Systemâ ‡439 Eyes: The Photoreceptor Systemâ ‡ 490 Testesâ ‡439 Ears: The Vestibuloauditory Systemâ ‡ 509 Intratesticular Ductsâ ‡ 449 Summary of Key Pointsâ ‡ 522 Excretory Genital Ductsâ ‡ 450 Assess Your Knowledgeâ ‡ 522 Accessory Glandsâ ‡ 451 Penisâ ‡456 APPENDIXâ ‡525 Summary of Key Pointsâ ‡ 457 FIGURE CREDITSâ ‡527 Assess Your Knowledgeâ ‡ 459 INDEXâ ‡529 Preface With this 14th edition, Junqueira’s Basic Histology continues as throughout the book as needed, and again make up a complete the preeminent source of concise yet thorough information atlas of cell, tissue, and organ structures fully compatible on human tissue structure and function. For nearly 45 years with the students’ own collection of glass or digital slides. A this educational resource has met the needs of learners for a virtual microscope with over 150 slides of all human tissues well-organized and concise presentation of cell biology and and organs is available: http://medsci.indiana.edu/junqueira/ histology that integrates the material with that of biochemistry, virtual/junqueira.htm. immunology, endocrinology, and physiology and provides As with the previous edition, the book facilitates learning an excellent foundation for subsequent studies in pathology. by its organization: The text is prepared specifically for students of medicine and other health-related professions, as well as for advanced An opening chapter reviews the histological techniques that allow understanding of cell and tissue structure. undergraduate courses in tissue biology. As a result of its value and appeal to students and instructors alike, Junqueira’s Basic Two chapters then summarize the structural and functional organization of human cell biology, Histology has been translated into a dozen different languages presenting the cytoplasm and nucleus separately. and is used by medical students throughout the world. This edition now includes with each chapter a set of The next seven chapters cover the four basic tissues that make up our organs: epithelia, connective tissue (and its multiple-choice Self-Test Questions that allow readers to assess major sub-types), nervous tissue, and muscle. their comprehension and knowledge of important material in that chapter. At least a few questions in each set utilize clinical Remaining chapters explain the organization and functional significance of these tissues in each of vignettes or cases to provide context for framing the medical the body’s organ systems, closing with up-to-date relevance of concepts in basic science, as recommended by consideration of cells in the eye and ear. the US National Board of Medical Examiners. As with the last edition, each chapter also includes a Summary of Key For additional review of what’s been learned or to Points designed to guide the students concerning what is assist rapid assimilation of the material in Junqueira’s Basic clearly important and what is less so. Summary Tables in Histology, McGraw-Hill has published a set of 200 full-color each chapter organize and condense important information, Basic Histology Flash Cards, Anthony Mescher author. Each further facilitating efficient learning. card includes images of key structures to identify, a summary Each chapter has been revised and shortened, while of important facts about those structures, and a clinical coverage of specific topics has been expanded as needed. Study comment. This valuable learning aid is available as a set of is facilitated by modern page design. Inserted throughout each actual cards from Amazon.com, or as an app for smart phones chapter are more numerous, short paragraphs that indicate how or tablets from the online App Store. the information presented can be used medically and which With its proven strengths and the addition of new features, emphasize the foundational relevance of the material learned. I am confident that Junqueira’s Basic Histology will continue The art and other figures are presented in each chapter, as one of the most valuable and most widely read educational with the goal to simplify learning and integration with resources in histology. Users are invited to provide feedback related material. The McGraw-Hill medical illustrations, now to the author with regard to any aspect of the book’s features. used throughout the text and supplemented by numerous animations in the electronic version of the text, are the Anthony L. Mescher most useful, thorough, and attractive of any similar medical Indiana University School of Medicine textbook. Electron and light micrographs have been replaced [email protected] ix Acknowledgments I wish to thank the students at Indiana University School of these valuable resources is gratefully acknowledged. Students are Medicine and the undergraduates at Indiana University with referred to those review books for hundreds of additional self- whom I have studied histology and cell biology for over 30 years assessment questions. and from whom I have learned much about presenting basic I am also grateful to my colleagues and reviewers from concepts most effectively. Their input has greatly helped in the throughout the world who provided specialized expertise task of maintaining and updating the presentations in this classic or original photographs, which are also acknowledged in textbook. As with the last edition the help of Sue Childress and figure captions. I thank those professors and students in the Dr. Mark Braun was invaluable in slide preparation and the United States, as well as Argentina, Canada, Iran, Ireland, Italy, virtual microscope for human histology respectively. Pakistan, and Syria, who provided useful suggestions that A major change in this edition is the inclusion of self- have improved the new edition of Junqueira’s Basic Histology. assessment questions with each topic/chapter. Many of these Finally, I am pleased to acknowledge the help and collegiality questions were used in my courses, but others are taken or provided by the staff of McGraw-Hill, especially editors modified from a few of the many excellent review books published Michael Weitz and Brian Kearns, whose work made possible by McGraw-Hill/Lange for students preparing to take the U.S. publication of this 14th edition of Junqueira’s Basic Histology. Medical Licensing Examination. These include Histology and Cell Biology: Examination and Board Review, by Douglas Paulsen; Anthony L. Mescher USMLE Road Map: Histology, by Harold Sheedlo; and Anatomy, Indiana University School of Medicine Histology, & Cell Biology: PreTest Self-Assessment & Review, by Robert Klein and George Enders. The use here of questions from [email protected] xi C H A P T E R 1 PREPARATION OF TISSUES FOR STUDY Histology & Its Methods of Study 1 AUTORADIOGRAPHY 9 Fixation 1 CELL & TISSUE CULTURE 10 Embedding & Sectioning 3 ENZYME HISTOCHEMISTRY 10 Staining 3 LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10 Bright-Field Microscopy 4 Immunohistochemistry 11 Fluorescence Microscopy 5 Hybridization Techniques 12 Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE Confocal Microscopy 5 SECTIONS 14 Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15 ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16 Transmission Electron Microscopy 8 Scanning Electron Microscopy 9 H istology is the study of the tissues of the body and how these tissues are arranged to constitute organs. This subject involves all aspects of tissue biology, with the focus on how cells’ structure and arrangement optimize functions specific to each organ. a better knowledge of tissue biology. Familiarity with the tools and methods of any branch of science is essential for a proper understanding of the subject. This chapter reviews common methods used to study cells and tissues, focusing on micro- scopic approaches. Tissues have two interacting components: cells and extracellular matrix (ECM). The ECM consists of many kinds of macromolecules, most of which form complex structures, such as collagen fibrils. The ECM supports the cells and ›â ºPREPARATION OF TISSUES contains the fluid transporting nutrients to the cells, and FOR STUDY carrying away their wastes and secretory products. Cells The most common procedure used in histologic research is produce the ECM locally and are in turn strongly influenced the preparation of tissue slices or “sections” that can be exam- by matrix molecules. Many matrix components bind to spe- ined visually with transmitted light. Because most tissues and cific cell surface receptors that span the cell membranes and organs are too thick for light to pass through, thin translu- connect to structural components inside the cells, forming a cent sections are cut from them and placed on glass slides for continuum in which cells and the ECM function together in microscopic examination of the internal structures. a well-coordinated manner. The ideal microscopic preparation is preserved so that the During development, cells and their associated matrix tissue on the slide has the same structural features it had in the become functionally specialized and give rise to fundamen- body. However, this is often not feasible because the prepara- tal types of tissues with characteristic structural features. tion process can remove cellular lipid, with slight distortions Organs are formed by an orderly combination of these tissues, of cell structure. The basic steps used in tissue preparation for and their precise arrangement allows the functioning of each light microscopy are shown in Figure 1–1. organ and of the organism as a whole. The small size of cells and matrix components makes his- tology dependent on the use of microscopes and molecular Fixation methods of study. Advances in biochemistry, molecular biol- To preserve tissue structure and prevent degradation by ogy, physiology, immunology, and pathology are essential for enzymes released from the cells or microorganisms, pieces of 1 2 CHAPTER 1â … â … Histology & Its Methods of Study FIGURE 1–1â ‡ Sectioning fixed and embedded tissue. 52°- 60°C (a) Fixation Dehydration Clearing Infiltration Embedding Drive wheel Block holder Paraffin block Tissue Steel knife b Most tissues studied histologically are prepared as shown, with Similar steps are used in preparing tissue for transmission elec- this sequence of steps (a): tron microscopy (TEM), except special fixatives and dehydrating solutions are used with smaller tissue samples and embedding ⌀ Fixation: Small pieces of tissue are placed in solutions of involves epoxy resins which become harder than paraffin to allow chemicals that cross-link proteins and inactivate degradative very thin sectioning. enzymes, which preserves cell and tissue structure. ⌀ Dehydration: The tissue is transferred through a series of (b) A microtome is used for sectioning paraffin-embedded tissues increasingly concentrated alcohol solutions, ending in 100%, for light microscopy. The trimmed tissue specimen is mounted which removes all water. in the paraffin block holder, and each turn of the drive wheel by ⌀ Clearing: Alcohol is removed in organic solvents in which the histologist advances the holder a controlled distance, gener- both alcohol and paraffin are miscible. ally from 1 to 10 μm. After each forward move, the tissue block ⌀ Infiltration: The tissue is then placed in melted paraffin until it passes over the steel knife edge and a section is cut at a thickness becomes completely infiltrated with this substance. equal to the distance the block advanced. The paraffin sections ⌀ Embedding: The paraffin-infiltrated tissue is placed in a small are placed on glass slides and allowed to adhere, deparaffinized, mold with melted paraffin and allowed to harden. and stained for light microscope study. For TEM, sections less than ⌀ Trimming: The resulting paraffin block is trimmed to expose 1 μm thick are prepared from resin-embedded cells using an the tissue for sectioning (slicing) on a microtome. ultramicrotome with a glass or diamond knife. organs are placed as soon as possible after removal from the microscopy, react with the amine groups (NH2) of proteins, body in solutions of stabilizing or cross-linking compounds preventing their degradation by common proteases. Glutaral- called fixatives. Because a fixative must fully diffuse through dehyde also cross-links adjacent proteins, reinforcing cell and the tissues to preserve all cells, tissues are usually cut into small ECM structures. fragments before fixation to facilitate penetration. To improve Electron microscopy provides much greater magnification cell preservation in large organs fixatives are often introduced and resolution of very small cellular structures and fixation via blood vessels, with vascular perfusion allowing fixation must be done very carefully to preserve additional “ultra- rapidly throughout the tissues. structural” detail. Typically in such studies glutaraldehyde- One widely used fixative for light microscopy is forma- treated tissue is then immersed in buffered osmium lin, a buffered isotonic solution of 37% formaldehyde. Both tetroxide, which preserves (and stains) cellular lipids as well this compound and glutaraldehyde, a fixative used for electron as proteins.  Preparation of Tissues for Study 3 Embedding & Sectioning Staining C H A P T E R To permit thin sectioning fixed tissues are infiltrated and Most cells and extracellular material are completely color- embedded in a material that imparts a firm consistency. less, and to be studied microscopically tissue sections must Embedding materials include paraffin, used routinely for light be stained (dyed). Methods of staining have been devised that microscopy, and plastic resins, which are adapted for both make various tissue components not only conspicuous but also light and electron microscopy. distinguishable from one another. Dyes stain material more or Before infiltration with such media the fixed tissue must less selectively, often behaving like acidic or basic compounds undergo dehydration by having its water extracted gradually and forming electrostatic (salt) linkages with ionizable radicals 1 by transfers through a series of increasing ethanol solutions, of macromolecules in tissues. Cell components such as nucleic Histology & Its Methods of Study â ‡ â ‡ Preparation of Tissues for Study ending in 100% ethanol. The ethanol is then replaced by an acids with a net negative charge (anionic) have an affinity for organic solvent miscible with both alcohol and the embedding basic dyes and are termed basophilic; cationic components, medium, a step referred to as clearing because infiltration with such as proteins with many ionized amino groups, stain more the reagents used here gives the tissue a translucent appearance. readily with acidic dyes and are termed acidophilic. The fully cleared tissue is then placed in melted paraffin Examples of basic dyes include toluidine blue, alcian blue, in an oven at 52°-60°C, which evaporates the clearing solvent and methylene blue. Hematoxylin behaves like a basic dye, and promotes infiltration of the tissue with paraffin, and then staining basophilic tissue components. The main tissue com- embedded by allowing it to harden in a small container of ponents that ionize and react with basic dyes do so because of paraffin at room temperature. Tissues to be embedded with acids in their composition (DNA, RNA, and glycosaminogly- plastic resin are also dehydrated in ethanol and then infiltrated cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain with plastic solvents that harden when cross-linking polymer- the acidophilic components of tissues such as mitochondria, izers are added. Plastic embedding avoids the higher tempera- secretory granules, and collagen. tures needed with paraffin, which helps avoid tissue distortion. Of all staining methods, the simple combination of The hardened block with tissue and surrounding embed- hematoxylin and eosin (H&E) is used most commonly. ding medium is trimmed and placed for sectioning in an Hematoxylin stains DNA in the cell nucleus, RNA-rich por- instrument called a microtome (Figure 1–1). Paraffin sections tions of the cytoplasm, and the matrix of cartilage, produc- are typically cut at 3-10 μm thickness for light microscopy, but ing a dark blue or purple color. In contrast, eosin stains other electron microscopy requires sections less than 1 μm thick. cytoplasmic structures and collagen pink (Figure 1–2a). Here One micrometer (1 μm) equals 1/1000 of a millimeter (mm) eosin is considered a counterstain, which is usually a single or 10–6 m. Other spatial units commonly used in microscopy dye applied separately to distinguish additional features of a are the nanometer (1 nm = 0.001 μm = 10–6 mm = 10–9 m) and tissue. More complex procedures, such as trichrome stains (eg, angstrom (1 Å = 0.1 nm or 10–4 μm). The sections are placed Masson trichrome), allow greater distinctions among various on glass slides and stained for light microscopy or on metal extracellular tissue components. grids for electron microscopic staining and examination. The periodic acid-Schiff (PAS) reaction utilizes the hexose rings of polysaccharides and other carbohydrate-rich › ⠺⠺ MEDICAL APPLICATION tissue structures and stains such macromolecules distinctly purple or magenta. Figure 1–2b shows an example of cells with Biopsies are tissue samples removed during surgery or rou- carbohydrate-rich areas well-stained by the PAS reaction. The tine medical procedures. In the operating room, biopsies DNA of cell nuclei can be specifically stained using a modifica- are fixed in vials of formalin for processing and microscopic tion of the PAS procedure called the Feulgen reaction. analysis in a pathology laboratory. If results of such analyses Basophilic or PAS-positive material can be further identi- are required before the medical procedure is completed, for fied by enzyme digestion, pretreatment of a tissue section with example to know whether a growth is malignant before the an enzyme that specifically digests one substrate. For example, patient is closed, a much more rapid processing method is pretreatment with ribonuclease will greatly reduce cytoplas- used. The biopsy is rapidly frozen in liquid nitrogen, preserv- mic basophilia with little overall effect on the nucleus, indicat- ing cell structures and making the tissue hard and ready for ing the importance of RNA for the cytoplasmic staining. sectioning. A microtome called a cryostat in a cabinet at Lipid-rich structures of cells are revealed by avoiding the subfreezing temperature is used to section the block with processing steps that remove lipids, such as treatment with tissue, and the frozen sections are placed on slides for rapid heat and organic solvents, and staining with lipid-soluble staining and microscopic examination by a pathologist. dyes such as Sudan black, which can be useful in diagnosis Freezing of tissues is also effective in histochemical stud- of metabolic diseases that involve intracellular accumulations ies of very sensitive enzymes or small molecules because of cholesterol, phospholipids, or glycolipids. Less common freezing, unlike fixation, does not inactivate most enzymes. methods of staining can employ metal impregnation tech- Finally, because clearing solvents often dissolve cell lipids in niques, typically using solutions of silver salts to visual certain fixed tissues, frozen sections are also useful when structures ECM fibers and specific cellular elements in nervous tissue. containing lipids are to be studied histologically. The Appendix lists important staining procedures used for most of the light micrographs in this book. 4 CHAPTER 1â … â … Histology & Its Methods of Study FIGURE 1–2â ‡ Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. G G G L L G G G a b Micrographs of epithelium lining the small intestine, (a) stained lumen, where projecting microvilli have a prominent layer of with H&E, and (b) stained with the PAS reaction for glycoproteins. glycoproteins at the lumen (L) and in the mucin-rich secretory With H&E, basophilic cell nuclei are stained purple while cyto- granules of goblet cells. Cell surface glycoproteins and mucin are plasm stains pink. Cell regions with abundant oligosaccharides PAS-positive because of their high content of oligosaccharides on glycoproteins, such as the ends of the cells at the lumen (L) and polysaccharides respectively. The PAS-stained tissue was or the scattered mucus-secreting goblet cells (G), are poorly counterstained with hematoxylin to show the cell nuclei. (a. X400; stained. With PAS, however, cell staining is most intense at the b. X300) Slide preparation, from tissue fixation to observation (or ocular lens) further magnifying this image and projecting with a light microscope, may take from 12 hours to 2½ days, it onto the viewer’s retina or a charge-coupled device (CCD) depending on the size of the tissue, the embedding medium, highly sensitive to low light levels with a camera and monitor. and the method of staining. The final step before microscopic The total magnification is obtained by multiplying the magni- observation is mounting a protective glass coverslip on the fying power of the objective and ocular lenses. slide with clear adhesive. The critical factor in obtaining a crisp, detailed image with a light microscope is its resolving power, defined as the smallest distance between two structures at which they can be ›â ºLIGHT MICROSCOPY seen as separate objects. The maximal resolving power of the light microscope is approximately 0.2 μm, which can permit Conventional bright-field microscopy, as well as more special- clear images magnified 1000-1500 times. Objects smaller or ized applications like fluorescence, phase-contrast, confocal, thinner than 0.2 μm (such as a single ribosome or cytoplasmic and polarizing microscopy, are all based on the interaction of microfilament) cannot be distinguished with this instrument. light with tissue components and are used to reveal and study Likewise, two structures such as mitochondria will be seen as tissue features. only one object if they are separated by less than 0.2 μm. The microscope’s resolving power determines the quality of the Bright-Field Microscopy image, its clarity and richness of detail, and depends mainly on With the bright-field microscope stained tissue is examined the quality of its objective lens. Magnification is of value only with ordinary light passing through the preparation. As shown when accompanied by high resolution. Objective lenses pro- in Figure 1–3, the microscope includes an optical system and viding higher magnification are designed to also have higher mechanisms to move and focus the specimen. The optical resolving power. The eyepiece lens only enlarges the image components are the condenser focusing light on the object obtained by the objective and does not improve resolution. to be studied; the objective lens enlarging and projecting the Virtual microscopy, typically used for study of bright- image of the object toward the observer; and the eyepiece field microscopic preparations, involves the conversion of a  Light Microscopy 5 FIGURE 1–3â ‡ Components and light path of a Fluorescence Microscopy C H A P T E R bright-field microscope. When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wave- Eyepiece Interpupillar adjustment length—a phenomenon called fluorescence. In fluores- Binocular tubes Head cence microscopy, tissue sections are usually irradiated with ultraviolet (UV) light and the emission is in the visible portion of the spectrum. The fluorescent substances appear bright on Stand a dark background. For fluorescent microscopy the instru- 1 Measuring ment has a source of UV or other light and filters that select Histology & Its Methods of Study â ‡ â ‡ Light Microscopy graticule Beamsplitter rays of different wavelengths emitted by the substances to be Revolving nosepiece visualized. Specimen Objective Fluorescent compounds with affinity for specific cell holder macromolecules may be used as fluorescent stains. Acridine Mechanical stage orange, which binds both DNA and RNA, is an example. On/off switch When observed in the fluorescence microscope, these nucleic Condenser Illumination intensity acids emit slightly different fluorescence, allowing them to be Field lens control localized separately in cells (Figure 1–4a). Other compounds Field such as DAPI and Hoechst stain specifically bind DNA and diaphragm Collector are used to stain cell nuclei, emitting a characteristic blue fluo- lens rescence under UV. Another important application of fluores- cence microscopy is achieved by coupling compounds such as X-Y Base translation fluorescein to molecules that will specifically bind to certain Tungsten mechanism cellular components and thus allow the identification of these halogen lamp structures under the microscope (Figure 1–4b). Antibodies labeled with fluorescent compounds are extremely important Photograph of a bright-field light microscope showing its in immunohistologic staining. (See the section Visualizing mechanical components and the pathway of light from the Specific Molecules.) substage lamp to the eye of the observer. The optical system has three sets of lenses: ⌀ The condenser collects and focuses a cone of light that illu- Phase-Contrast Microscopy minates the tissue slide on the stage. Unstained cells and tissue sections, which are usually trans- ⌀ Objective lenses enlarge and project the illuminated parent and colorless, can be studied with these modified image of the object toward the eyepiece. Interchangeable light microscopes. Cellular detail is normally difficult to see objectives with different magnifications routinely used in histology include X4 for observing a large area (field) of the in unstained tissues because all parts of the specimen have tissue at low magnification; X10 for medium magnification roughly similar optical densities. Phase-contrast micros- of a smaller field; and X40 for high magnification of more copy, however, uses a lens system that produces visible images detailed areas. from transparent objects and, importantly, can be used with ⌀ The two eyepieces or oculars magnify this image another living, cultured cells (Figure 1–5). X10 and project it to the viewer, yielding a total magnifica- tion of X40, X100, or X400. Phase-contrast microscopy is based on the principle that light changes its speed when passing through cellular (Used with permission from Nikon Instruments.) and extracellular structures with different refractive indices. These changes are used by the phase-contrast system to cause the structures to appear lighter or darker in relation to each stained tissue preparation to high-resolution digital images other. Because they allow the examination of cells without and permits study of tissues using a computer or other digi- fixation or staining, phase-contrast microscopes are promi- tal device, without an actual stained slide or a microscope. In nent tools in all cell culture laboratories. A modification of this technique regions of a glass-mounted specimen are cap- phase-contrast microscopy is differential interference tured digitally in a grid-like pattern at multiple magnifications microscopy with Nomarski optics, which produces an image using a specialized slide-scanning microscope and saved as of living cells with a more apparent three-dimensional (3D) thousands of consecutive image files. Software then converts aspect (Figure 1–5c). this dataset for storage on a server using a format that allows access, visualization, and navigation of the original slide with common web browsers or other devices. With advantages in Confocal Microscopy cost and ease of use, virtual microscopy is rapidly replacing With a regular bright-field microscope, the beam of light is rel- light microscopes and collections of glass slides in histology atively large and fills the specimen. Stray (excess) light reduces laboratories for students. contrast within the image and compromises the resolving 6 CHAPTER 1â … â … Histology & Its Methods of Study FIGURE 1–4â ‡ Appearance of cells with fluorescent microscopy. N N R a b Components of cells are often stained with compounds visible by filaments show nuclei with blue fluorescence and actin filaments fluorescence microscopy. stained green. Important information such as the greater density (a) Acridine orange binds nucleic acids and causes DNA in cell of microfilaments at the cell periphery is readily apparent. (Both nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to X500) appear orange in these cells of a kidney tubule. (Figure 1–4b, used with permission from Drs Claire E. Walczak and Rania Rizk, Indiana University School of Medicine, (b) Cultured cells stained with DAPI (4′,6-diamino-2-phenylindole) Bloomington.) that binds DNA and with fluorescein-phalloidin that binds actin FIGURE 1–5â ‡ Unstained cells’ appearance in three types of light microscopy. a b c Living neural crest cells growing in culture appear differently in-phase light differently and produce an image of these features with various techniques of light microscopy. Here the same field in all the cells. of unstained cells, including two differentiating pigment cells, is (c) Differential interference microscopy: Cellular details are shown using three different methods (all X200): highlighted in a different manner using Nomarski optics. Phase- (a) Bright-field microscopy: Without fixation and staining, only contrast microscopy, with or without differential interference, is the two pigment cells can be seen. widely used to observe live cells grown in tissue culture. (b) Phase-contrast microscopy: Cell boundaries, nuclei, and (Used with permission from Dr Sherry Rogers, Department of Cell cytoplasmic structures with different refractive indices affect Biology and Physiology, University of New Mexico, Albuquerque, NM.)  Light Microscopy 7 the specimen allows them to be digitally reconstructed into a FIGURE 1–6â ‡ Principle of confocal microscopy. 3D image. C H A P T E R Laser Polarizing Microscopy Polarizing microscopy allows the recognition of stained or unstained structures made of highly organized subunits. When normal light passes through a polarizing filter, it exits 1 Scanner vibrating in only one direction. If a second filter is placed in Histology & Its Methods of Study â ‡ â ‡ Light Microscopy the microscope above the first one, with its main axis per- Detector pendicular to the first filter, no light passes through. If, how- ever, tissue structures containing oriented macromolecules are located between the two polarizing filters, their repeti- tive structure rotates the axis of the light emerging from the polarizer and they appear as bright structures against a dark background (Figure 1–7). The ability to rotate the direction of vibration of polarized light is called birefringence and is Plate with pinhole Beam splitter FIGURE 1–7â ‡ Tissue appearance with bright-field and polarizing microscopy. Lens Other Focal plane out-of-focus Specimen planes Although a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the blind. Thus, only one very thin plane of the specimen is focused at a a time. The diagram shows the practical arrangement of a confo- cal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pin- hole and a detector. Light from components of the specimen that are above or below the focused plane is blocked by the blind. The laser scans the specimen so that a larger area of the specimen can be observed. power of the objective lens. Confocal microscopy (Figure 1–6) avoids these problems and achieves high resolution and sharp focus by using (1) a small point of high-intensity light, often from a laser, and (2) a plate with a pinhole aperture in front of the image detector. The point light source, the focal point of b the lens, and the detector’s pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (confo- Polarizing light microscopy produces an image only of material cal), and unfocused light does not pass through the pinhole. having repetitive, periodic macromolecular structure; features This greatly improves resolution of the object in focus and without such structure are not seen. Pieces of thin, unsec- allows the localization of specimen components with much tioned mesentery were stained with red picrosirius, orcein, and greater precision than with the bright-field microscope. hematoxylin, placed on slides and observed by bright-field (a) Confocal microscopes include a computer-driven mirror and polarizing (b) microscopy. system (the beam splitter) to move the point of illumination (a) With bright-field microscopy collagen fibers appear red, with thin elastic fibers and cell nuclei darker. (X40) across the specimen automatically and rapidly. Digital images captured at many individual spots in a very thin plane of focus (b) With polarizing microscopy, only the collagen fibers are vis- ible and these exhibit intense yellow or orange birefringence. are used to produce an “optical section” of that plane. Creat- (a: X40; b: X100) ing such optical sections at a series of focal planes through 8 CHAPTER 1â … â … Histology & Its Methods of Study a feature of crystalline substances or substances containing The wavelength in an electron beam is much shorter than that highly oriented molecules, such as cellulose, collagen, micro- of light, allowing a 1000-fold increase in resolution. tubules, and actin filaments. The utility of all light microscopic methods is greatly Transmission Electron Microscopy extended through the use of digital cameras. Many features The transmission electron microscope (TEM) is an imag- of digitized histological images can be analyzed quantitatively ing system that permits resolution around 3 nm. This high using appropriate software. Such images can also be enhanced resolution allows isolated particles magnified as much as to allow objects not directly visible through the eyepieces to be 400,000 times to be viewed in detail. Very thin (40-90 nm), examined on a monitor. resin-embedded tissue sections are typically studied by TEM at magnifications up to approximately 120,000 times. ›â ºELECTRON MICROSCOPY Figure 1–8a indicates the components of a TEM and the basic principles of its operation: a beam of electrons focused Transmission and scanning electron microscopes are based on using electromagnetic “lenses” passes through the tissue sec- the interaction of tissue components with beams of electrons. tion to produce an image with black, white, and intermediate FIGURE 1–8â ‡ Electron microscopes. Electron gun Electron gun Cathode Cathode 3 mm Anode Anode Copper grid Condensor lens with three sections Lens Specimen Column Objective lens holder Lens Column Scanner Intermediate lens Electron detector TEM image Lens SEM image Projector lens Image on viewing screen Specimen Electron detector with CCD camera (a) Transmission electron microscope (b) Scanning electron microscope Electron microscopes are large instruments generally housed in a In a TEM image areas of the specimen through which electrons specialized EM facility. passed appear bright (electron lucent), while denser areas or (a) Schematic view of the major components of a transmission elec- those that bind heavy metal ions during specimen preparation tron microscope (TEM), which is configured rather like an upside- absorb or deflect electrons and appear darker (electron dense). down light microscope. With the microscope column in a vacuum, a Such images are therefore always black, white, and shades of gray. metallic (usually tungsten) filament (cathode) at the top emits elec- (b) The scanning electron microscope (SEM) has many similarities trons that travel to an anode with an accelerating voltage between to a TEM. However, here the focused electron beam does not pass 60 and 120 kV. Electrons passing through a hole in the anode form a through the specimen, but rather is moved sequentially (scanned) beam that is focused electromagnetically by circular electric coils from point to point across its surface similar to the way an electron in a manner analogous to the effect of optical lenses on light. beam is scanned across a television tube or screen. For SEM speci- The first lens is a condenser focusing the beam on the sec- mens are coated with metal atoms with which the electron beam tion. Some electrons interact with atoms in the section, being interacts, producing reflected electrons and newly emitted secondary absorbed or scattered to different extents, while others are simply electrons. All of these are captured by a detector and transmitted to transmitted through the specimen with no interaction. Electrons amplifiers and processed to produce a black-and-white image on the reaching the objective lens form an image that is then magnified monitor. The SEM shows only surface views of the coated specimen and finally projected on a fluorescent screen or a charge-coupled but with a striking 3D, shadowed quality. The inside of organs or cells device (CCD) monitor and camera. can be analyzed after sectioning to expose their internal surfaces.  Autoradiography 9 shades of gray regions. These regions of an electron micro- layer of heavy metal (often gold) which reflects electrons in graph correspond to tissue areas through which electrons a beam scanning the specimen. The reflected electrons are C H A P T E R passed readily (appearing brighter or electron-lucent) and captured by a detector, producing signals that are processed areas where electrons were absorbed or deflected (appearing to produce a black-and-white image. SEM images are usually darker or more electron-dense). To improve contrast and reso- easy to interpret because they present a three-dimensional lution in TEM, compounds with heavy metal ions are often view that appears to be illuminated in the same way that large added to the fixative or dehydrating solutions used for tissue objects are seen with highlights and shadows caused by light. preparation. These include osmium tetroxide, lead citrate, 1 and uranyl compounds, which bind cellular macromolecules, ›â ºAUTORADIOGRAPHY Histology & Its Methods of Study â ‡ â ‡Autoradiography increasing their electron density and visibility. Cryofracture and freeze etching are techniques that Microscopic autoradiography is a method of localizing allow TEM study of cells without fixation or embedding and newly synthesized macromolecules in cells or tissue sections. have been particularly useful in the study of membrane struc- Radioactively labeled metabolites (nucleotides, amino acids, ture. In these methods very small tissue specimens are rap- sugars) provided to the living cells are incorporated into spe- idly frozen in liquid nitrogen and then cut or fractured with a cific macromolecules (DNA, RNA, protein, glycoproteins, and knife. A replica of the frozen exposed surface is produced in a polysaccharides) and emit weak radiation that is restricted vacuum by applying thin coats of vaporized platinum or other to those regions where the molecules are located. Slides with metal atoms. After removal of the organic material, the replica radiolabeled cells or tissue sections are coated in a darkroom of the cut surface can be examined by TEM. With membranes with photographic emulsion in which silver bromide crystals the random fracture planes often split the lipid bilayers, expos- ing protein components whose size, shape, and distribution act as microdetectors of the radiation in the same way that are difficult to study by other methods. they respond to light in photographic film. After an adequate exposure time in lightproof boxes, the slides are developed photographically. Silver bromide crystals reduced by the radia- Scanning Electron Microscopy tion produce small black grains of metallic silver, which under Scanning electron microscopy (SEM) provides a high- either the light microscope or TEM indicate the locations of resolution view of the surfaces of cells, tissues, and organs. Like radiolabeled macromolecules in the tissue (Figure 1–9). the TEM, this microscope produces and focuses a very narrow Much histological information becomes available by beam of electrons, but in this instrument the beam does not autoradiography. If a radioactive precursor of DNA (such pass through the specimen (Figure 1–8b). Instead, the surface as tritium-labeled thymidine) is used, it is possible to know of the specimen is first dried and spray-coated with a very thin which cells in a tissue (and how many) are replicating DNA FIGURE 1–9â ‡ Microscopic autoradiography. L G G a b Autoradiographs are tissue preparations in which particles called (a) Black grains of silver from the light-sensitive material coating silver grains indicate the cells or regions of cells in which specific the specimen are visible over cell regions with secretory granules macromolecules were synthesized just prior to fixation. Shown and the duct indicating glycoprotein locations. (X1500) here are autoradiographs from the salivary gland of a mouse (b) The same tissue prepared for TEM autoradiography shows sil- injected with 3H-fucose 8 hours before tissue fixation. Fucose was ver grains with a coiled or amorphous appearance again localized incorporated into oligosaccharides, and the free 3H-fucose was mainly over the granules (G) and in the gland lumen (L). (X7500) removed during fixation and sectioning of the gland. Autoradio- (Figure 1–9b, used with permission from Drs Ticiano G. Lima and graphic processing and microscopy reveal locations of newly syn- A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.) thesized glycoproteins containing that sugar. 10 CHAPTER 1â … â … Histology & Its Methods of Study and preparing to divide. Dynamic events may also be analyzed. For example, if one wishes to know where in the cell protein is ›â ºENZYME HISTOCHEMISTRY produced, if it is secreted, and its path in the cell before being Enzyme histochemistry (or cytochemistry) is a method for secreted, several animals are injected with a radioactive amino localizing cellular structures using a specific enzymatic activ- acid and tissues collected at different times after the injections. ity present in those structures. To preserve the endogenous Autoradiography of the tissues from the sequential times will enzymes histochemical procedures usually use unfixed or indicate the migration of the radioactive proteins. mildly fixed tissue, which is sectioned on a cryostat to avoid adverse effects of heat and organic solvents on enzymatic ›â ºCELL & TISSUE CULTURE activity. For enzyme histochemistry (1) tissue sections are immersed in a solution containing the substrate of the enzyme Live cells and tissues can be maintained and studied outside to be localized; (2) the enzyme is allowed to act on its sub- the body in culture (in vitro). In the organism (in vivo), cells strate; (3) the section is then put in contact with a marker are bathed in fluid derived from blood plasma and containing compound that reacts with a product of the enzymatic action many different molecules required for survival and growth. on the substrate; and (4) the final product from the marker, Cell culture allows the direct observation of cellular behavior which must be insoluble and visible by light or electron under a phase-contrast microscope and many experiments microscopy, precipitates over the site of the enzymes, identify- technically impossible to perform in the intact animal can be ing their location. accomplished in vitro. Examples of enzymes that can be detected histochemi- The cells and tissues are grown in complex solutions of cally include the following: known composition (salts, amino acids, vitamins) to which ⌀ Phosphatases, which remove phosphate groups from serum or specific growth factors are added. Cells to be cultured macromolecules (Figure 1–10). are dispersed mechanically or enzymatically from a tissue or ⌀ Dehydrogenases, which transfer hydrogen ions from organ and placed with sterile procedures in a clear dish to one substrate to another, such as many enzymes of the which they adhere, usually as a single layer (Figure 1–5). Such citric acid (Krebs) cycle, allowing histochemical identifi- preparations are called primary cell cultures. Some cells can cation of such enzymes in mitochondria. be maintained in vitro for long periods because they become ⌀ Peroxidase, which promotes the oxidation of sub- immortalized and constitute a permanent cell line. Most cells strates with the transfer of hydrogen ions to hydrogen obtained from normal tissues have a finite, genetically pro- peroxide. grammed life span. However certain changes (some related to oncogenes; see Chapter 3) can promote cell immortality, a process called transformation, and are similar to the initial › ⠺⠺ MEDICAL APPLICATION changes in a normal cell’s becoming a cancer cell. Improve- Many enzyme histochemical procedures are used in the ments in culture technology and use of specific growth factors medical laboratory, including Perls’ Prussian blue reaction for now allow most cell types to be maintained in vitro. iron (used to diagnose the iron storage diseases, hemochro- As shown in Chapter 2, incubation of living cells in vitro matosis and hemosiderosis), the PAS-amylase and alcian blue with a variety of new fluorescent compounds that are seques- reactions for polysaccharides (to detect glycogenosis and tered and metabolized in specific compartments of the cell mucopolysaccharidosis), and reactions for lipids and sphin- provides a new approach to understanding these compart- golipids (to detect sphingolipidosis). ments both structurally and physiologically. Other histologic techniques applied to cultured cells have been particularly important for understanding the locations and functions of microtubules, microfilaments, and other components of the cytoskeleton. ›â ºVISUALIZING SPECIFIC MOLECULES A specific macromolecule present in a tissue section may also be identified by using tagged compounds or macromolecules › ⠺⠺ MEDICAL APPLICATION that bind specifically with the molecule of interest. The com- Cell culture is very widely used to study molecular changes pounds that interact with the molecule must be visible with that occur in cancer; to analyze infectious viruses, myco- the light or electron microscope, often by being tagged with a plasma, and some protozoa; and for many routine genetic or detectible label. The most commonly used labels are fluores- chromosomal analyses. Cervical cancer cells from a patient cent compounds, radioactive atoms that can be detected with later identified as Henrietta Lacks, who died from the disease autoradiography, molecules of peroxidase or other enzymes in 1951, were used to establish one of the first cell lines, that can be detected with histochemistry, and metal (usually called HeLa cells, which are still used in research on cellular gold) particles that can be seen with light and electron micros- structure and function throughout the world. copy. These methods can be used to detect and localize specific sugars, proteins, and nucleic acids.  Visualizing Specific Molecules 11 Examples of molecules that interact specifically with FIGURE 1–10â ‡ Enzyme histochemistry. other molecules include the following: C H A P T E R ⌀ Phalloidin, a compound extracted from mushroom, Amanita phalloides, interacts strongly with the actin pro- tein of microfilaments. L ⌀ Protein A, purified from Staphylococcus aureus bacte- ria, binds to the Fc region of antibody molecules, and can therefore be used to localize naturally occurring or 1 applied antibodies bound to cell structures. Histology & Its Methods of Study â ‡ â ‡ Visualizing Specific Molecules ⌀ Lectins, glycoproteins derived mainly from plant seeds, bind to carbohydrates with high affinity and specificity. Different lectins bind to specific sugars or sequences of sugar residues, allowing fluorescently labeled lectins to be used to stain specific glycoproteins or other macro- molecules bearing specific sequences of sugar residues. Immunohistochemistry A highly specific interaction between macromolecules is that between an antigen and its antibody. For this reason labeled antibodies are routinely used in immunohistochemistry L L to identify and localize many specific proteins, not just those with enzymatic activity that can be demonstrated by histochemistry. aa The body’s immune cells interact with and produce anti- bodies against other macromolecules—called antigens—that are recognized as “foreign,” not a normal part of the organism, and potentially dangerous. Antibodies belong to the immu- noglobulin family of glycoproteins and are secreted by lym- phocytes. These molecules normally bind specifically to their Ly provoking antigens and help eliminate them. Widely applied for both research and diagnostic pur- poses, every immunohistochemical technique requires an antibody against the protein that is to be detected. This means Ly that the protein must have been previously purified using bio- chemical or molecular methods so that antibodies against it can be produced. To produce antibodies against protein x of a certain animal species (eg, a human or rat), the isolated pro- tein is injected into an animal of another species (eg, a rabbit or a goat). If the protein’s amino acid sequence is sufficiently b N different for this animal to recognize it as foreign—that is, as an antigen—the animal will produce antibodies against the protein. (a) Micrograph of cross sections of kidney tubules treated Different groups (clones) of lymphocytes in the injected histochemically to demonstrate alkaline phosphatases (with animal recognize different parts of protein x and each clone maximum activity at an alkaline pH) showing strong activity of produces an antibody against that part. These antibodies are this enzyme at the apical surfaces of the cells at the lumens (L) of the tubules. (X200) collected from the animal’s plasma and constitute a mixture of polyclonal antibodies, each capable of binding a different (b) TEM image of a kidney cell in which acid phosphatase has been localized histochemically in three lysosomes (Ly) near the region of protein x. nucleus (N). The dark material within these structures is lead It is also possible, however, to inject protein x into a phosphate that precipitated in places with acid phosphatase mouse and a few days later isolate the activated lymphocytes activity. (X25,000) and place them into culture. Growth and activity of these cells (Figure 1–10b, used with permission from Dr Eduardo can be prolonged indefinitely by fusing them with lymphocytic Katchburian, Department of Morphology, Federal University of São Paulo, Brazil.) tumor cells to produce hybridoma cells. Different hybridoma clones produce different antibodies against the several parts 12 CHAPTER 1â … â … Histology & Its Methods of Study of protein x and each clone can be isolated and cultured sepa- The indirect method is used more widely in research and rately so that the different antibodies against protein x can be pathologic tests because it is more sensitive, with the extra collected separately. Each of these antibodies is a monoclo- level of antibody binding serving to amplify the visible signal. nal antibody. An advantage to using a monoclonal antibody Moreover, the same preparation of labeled secondary antibody rather than polyclonal antibodies is that it can be selected to can be used in studies with different primary antibodies (spe- be highly specific and to bind strongly to the protein to be cific for different antigens) as long as all these are made in the detected, with less nonspecific binding to other proteins that same species. There are other indirect methods that involve the are similar to the one of interest. use of other intermediate molecules, such as the biotin-avidin In immunohistochemistry a tissue section that one technique, which are also used to amplify detection signals. believes contains the protein of interest is incubated in a solu- Examples of indirect immunocytochemistry are shown in tion containing antibody (either monoclonal or polyclonal) Figure 1–12, demonstrating the use of this method with cells against this protein. The antibody binds specifically to the in culture or after tissue sectioning for both light microscopy protein and after a rinse the protein’s location in the tissue or and TEM. cells can be seen with either the light or electron microscope by visualizing the antibody. Antibodies are commonly tagged › ⠺⠺ MEDICAL APPLICATION with fluorescent compounds, with peroxidase or alkaline Because cells in some diseases, including many cancer cells, phosphatase for histochemical detection, or with electron- often produce proteins unique to their pathologic condition, dense gold particles for TEM. immunohistochemistry can be used by pathologists to diag- As Figure 1–11 indicates, there are direct and indirect nose many diseases, including certain types of tumors and methods of immunocytochemistry. The direct method just some virus-infected cells. Table 1-1 shows some applications involves a labeled antibody that binds the protein of interest. of immunocytochemistry routinely used in clinical practice. Indirect immunohistochemistry involves sequential application of two antibodies and additional washing steps. The (primary) antibody specifically binding the protein of interest Hybridization Techniques is not labeled. The detectible tag is conjugated to a second- Hybridization usually implies the specific binding between ary antibody made in an animal species different (“foreign”) two single strands of nucleic acid, which occurs under appro- from that which made the primary antibody. For example, pri- priate conditions if the strands are complementary. The greater mary antibodies made by mouse lymphocytes (such as most the similarities of their nucleotide sequences, the more read- monoclonal antibodies) are specifically recognized and bound ily the complementary strands form “hybrid” double-strand by antibodies made in a rabbit or goat injected with mouse molecules. Hybridization at stringent conditions allows the antibody immunoglobulin. specific identification of sequences in genes or RNA. This can FIGURE 1–11â ‡ Immunocytochemistry techniques. Labeled secondary Labeled Unlabeled antibody antibody primary antibody Antigen Antigen Tissue section Glass slide Direct Indirect Immunocytochemistry (or immunohistochemistry) can be direct labeled secondary antibody is obtained that was (1) made in or indirect. Direct immunocytochemistry (left) uses an antibody another species against immunoglobulin proteins (antibodies) made against the tissue protein of interest and tagged directly from the species in which the primary antibodies were made and with a label such as a fluorescent compound or peroxidase. When (2) labeled with a fluorescent compound or peroxidase. When placed with the tissue section on a slide, these labeled antibod- the labeled secondary antibody is applied to the tissue section, it ies bind specifically to the protein (antigen) against which they specifically binds the primary antibodies, indirectly labeling the were produced and can be visualized by the appropriate method. protein of interest on the slide. Because more than one labeled Indirect immunocytochemistry (right) uses first a primary secondary antibody can bind each primary antibody molecule, antibody ma