Methods in

IMMUNOLOGY

and IMMUNOCHEMISTRY

Edited by

C U R T I S A . W I L L I A M STHE ROCKEFELLER UNIVERSITY

NEW YORK, NEW YORK

M E R R I L L W . C H A S ETHE ROCKEFELLER UNIVERSITY FACHBEREICH BIOLOGIE (10)NEW YORK, NEW YORK der Technischcn Hcdischule Darmstadt

- B i b i i c - t h e k -

D - 6100 D a r u s I a d I / B. R. D.

Schniiispahnsi'rafie

Volume V

Antigen-Antibody Reactions inVivo Inv.-Nr.M v ^oSl__

1976

A C A D E M I C PRESS New York San Francisco LondonA SUBSIDIARY OF HARCOURT BRACE JOVANOVICH, PUBLISHERS

:Topical Listing of Contents

CONTRIBUTORS TO VOLUME V xvii

PREFACE xxi

CONTENTS OF OTHER VOLUMES xxiii

Chapter 19. Anaphylaxis

A. SYSTEMIC ANAPHYLAXIS 1

Active Anaphylaxis: Antigens, 4; Procedures with Guinea Pigs, 5, Mice, 6,Rats, 7, Rabbits, 9, Dogs, 10, Primates, 10, Chickens, 10.Passive Anaphylaxis: Principles, 11; Procedures with Various Species, 12.

B. LOCAL ANAPHYLAXIS 16

Active Cutaneous Anaphylaxis, 21.Passive Cutaneous Anaphylaxis, 16: General Procedures, 17; Special Proce-dures for PCA in Guinea Pigs, 18; Procedures for Mice, Rats, Rabbits, 21.Semiquantitative Passive Cutaneous Anaphylaxis, 22.Prausnitz-Kiistner Skin Tests in Man for Reagins (IgE Antibody), 27.Passive Transfer of Human Reagins into the Monkey, 31.

|C. ANAPHYLAXIS IN ISOLATED TISSUES AND CELLS . ' 42

1. Anaphylaxis in Isolated Tissues.Principles and Applications of the Schultz-Dale Reaction, 42.Anaphylactic Release of Histamine, 45, of Serotonin, 53, of SRS-A, 53, ofBradykinin, 56. Laboratory Procedures, 65; Methods of Active and PassiveSensitization, 74; Sensitization in Vitro, 81.The Quantitative Schultz-Dale Reaction, 83. \Chemical Estimation of Materials Released during Anaphylaxis, 87: Hista-mine, 90, Serotonin, 91. Experimental Protocol, 92.Applications and Limitations of Schultz-Dale Reactions, 94: Desensitizationand Specificity, 96; Competitive Sensitization, 99; Reverse Passive Sensitiza-tion by Antigens, 100.

2. Anaphylactic Responses with Isolated Lung Slices, 101.Preparation of Guinea Pig Lung Slices, 101; Challenge with Antigen, 104;Inhibition and Enhancement of Release of Mediators, 106.

Primate and Human Lung Fragments, 106.

VI CONTENTS

3. Anaphylactic Release of Histamine from Rat Mast Cells, 108.Collection Techniques, 108; Experimental Procedures, 111; Antibody-Medi-ated Release of Histamine: by IgE, 111, by IgG,,, 114.

4. Responses of Isolated Leukocytes and Platelets in Reaginic Allergy, 114;Human Leukocytes, 114; Rabbit Leukocytes, 120; Rabbit Platelets, 122;Histamine Release by Platelets: Due to Antigen-Antibody Reactions inPlasma, 122; Due to Reaction of Antigen and Sensitized Leukocytes, 125.

D. ASSAY OF PHARMACOLOGICALLY ACTIVE MATERIALS 126

1. Assay of Histamine, 126: Biological Assay, 126; Fluorometric Assay, 129;Enzymatic-Isotopic Microassay, 135.

2. Assay of Serotonin, 139: General Method, 141; Assay in Blood Platelets, 142;Indolealkylamine-Ninhydrin Fluorometric Method, 142; o-PhthalaldehydeFluorometric Method, 143; Enzymatic-Isotope Micromethod, 143; Radioim-munoassay, 144.

3. Biological Assay of Slow Reacting Substances—SRS-A, Bradykinin, Prosta-glandins, 145.System for Biological Assays, 146. Bioassays: for SRS-A, 147; for Bradykinin,148; for Prostaglandins, 149.

4. Immunoassay for Bradykinin and Kininogen, 149.Radioimmunoassay of Bradykinin, 149: Assay with Beta Isotopes and GelFiltration, 151; Assay with 1Z5I-Tyrosine-Bradykinin, 152; Collection andProcessing of Biological Fluids, 154.Radial Immunodiffusion Assay of Kininogen, 155.

Chapter 20. The Arthus and Related Reactions

A. CUTANEOUS ARTHUS REACTIONS 159

Active, Direct Passive, Reversed Passive Arthus Reactions, 159.Local Passive Arthus Reactions, 164; Reactions Induced by Soluble Com-plexes, 164.Tissues Employed in Arthus Studies, 164.Elicitation Requirement for Precipitating Antibody and for Complement Fixa-tion, 166.

B. METHODS OF MODIFYING ARTHUS REACTIONS 167

Elimination of Polymorphonuclear Leukocytes, 167.Depletion of Serum Complement, 169; Preparation of Cobra Venom Factor,169: Assay, 170; Administration, 171.

C. ARTHUS P H E N O M E N O N IN DISEASE PROCESSES 172

Serum Sickness Arteritis, 173; Acute Nephrotoxic Nephritis, 174; CutaneousReaction to Anti-Kidney Basement Membrane Antibody, 175.

Chapter 21. Analysis of Antigen-Antibody Reactions in Cornea

Techniques, 177; Local Antigen-Antibody Reactions in the Cornea, 178:Wessely Phenomenon, 179; Anaphylactic Keratitis, 180; Local IntracornealInjections in Rabbits with Both Antigen and Antibody, 182.

CONTENTS Vll

Chapter 22. Immunological Tolerance

A. INTRODUCTION > 185

B. TOLERANCE TO HETEROLOGOUS SERUM PROTEINS AND

RELATED SUBSTANCES 188

1. Induction in Neonatal Animals, 188. Rabbits: with Albumins and Globulins,188, with Hapten-Conjugates, 192, with Synthetic Polypeptides, 193; Inductionof Tolerance, 189; Immune Elimination Techniques, 189.Mice with Globulins and Albumins, 193; Chickens, 195; Guinea Pigs withProteins and Ovalbumin, 197; Rats, 201; Goats, 201.

2. Induction of Tolerance in Normal Adult Animals, 201. Rabbits, 202, 205.Mice with Bovine 7-Globulin, 203; with Monomeric Human 7-Globulin, 204;with Human 7-Globulin, 207; with Bovine Serum Albumin, 207; with Micro-bial Polysaccharides (Pneumococcal Capsular Polysaccharides, DetoxifiedLipopolysaccharide, Levan), 208. Preparation of Monomeric HGG, 204, ofAggregated HGG, 204. Testing for Tolerance: Plaque Assay with Spleen Cells,204; Transfer of Thymic or Bone-Marrow Cells, 205.

Guinea Pigs with Bovine 7-Globulin and Bovine Serum Albumin, 209. Chick-ens, 210.

3. Induction of Tolerance in Irradiated Normal Animals, 210.

C. TOLERANCE TO HOMOLOGOUS AND HETEROLOGOUS ERYTHROCYTES . 213

Chickens, 213; Rats, 214; Mice, 215.

D. INDUCTION OF TOLERANCE TO CHEMICAL H A P T E N S . . . . 215

Induction by Feeding, 217, bj- Injection, 219.

E. INDUCTION OF TOLERANCE in Vitro 221

Lipopolysaccharide, 221; Polymerized Flagellin, 221; Haptens, 222.

Chapter 23. Immune Suppression and Induction of Tolerance with Chemical

Agents

A. INTRODUCTION • . 225

B. BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES OF T H E AGENT . 226

6-Mercaptopurine, 226; Cyclophosphamide, 228; 5-Fluoro-2-deoxyuridine(FUDR), 228; Methotrexate, 229; Actinomycin D, 230.

C. IMMUNOSUPPRESSIVE PROPERTIES OF T H E AGENTS . . . . . 230

1. 6-Mercaptopurine: Antibody Synthesis Suppressed: in Rabbits, 230, in Mice,232.

2. Cyclophosphamide: Antibody Synthesis Suppressed in Guinea Pigs (Proteins),233, in Mice to Sheep and Horse Erythrocytes, 234.Induction of Delayed Hypersensitivity Suppressed in Guinea Pigs, 234.

3. Methotrexate: Suppression of Antibody and Delayed-Type Intradermal Re-sponses of Guinea Pigs to Ovalbumin, 235.

4. 5-Fluoro-2-deoxyuridine (FUDR) : Antibody Synthesis Suppressed in Miceby a Single Dose of FUDR, 236, by Multiple Doses, 237.

Vlll CONTENTS

5. Actinomycin D: Inhibition of Antibody Synthesis in Vitro by Spleen Cellsof Primed Rabbits, 237.

Chapter 24. Methods of Applications of Radiation in ImmunologicalResearch

A. RADIATION MEASUREMENTS 240

Characteristics of the X-Ray Beam, 240; Dosimetry, 241: Roentgen Units,242; Importance of Rate of Exposure, 242; Problems in Determining AbsorbedDose, 243; Electronic Equilibrium, 244.

B. REPRESENTATIVE E F F E C T S OF RADIATION 246

1. In Vitro Radiation Effects on Serum, 247.2. Total Body Irradiation, 247.

Animal Preparation, 247; LD'uo Single Doses for Various Species, 247; Dif-ferential Effects on White Cells of Various Species, 248.Effects on Antibody Formation, 250; on Latent Period and Peak Titer, 251;on Dose Effects, 251, 252.Effect of Antigen Dose at Fixed Irradiation Dose, 252.Restoration of X-Ray Depression by Normal Cells or Nucleic Acid Deriva-tives, 253.Delayed Responses within Irradiated Groups of Animals, 253; "Relative Im-mune State" as Measure of Radiation Injury, 253.Antibody Synthesis by Irradiated Cells Transplanted into Heavily IrradiatedAnimals, 254.

3. Low Level Protracted Semicontinuous Radiation, 254.4. Partial Body Radiation: Selective Shielding, 255.

Lead Shields for Spleen-Appendix: Shielding from or Selective Exposure toX-Ray, 255; Shields for Liver, 256; Shielding and Irradiating of Legs, 256;Practical Examples of Selective Shielding, 257.

C. CONCLUDING REMARKS 258

Precautions—Depression or Enhancement of Antibody Synthesis by X-Ray Ir-radiation—Chimeras Produced by Irradiation—Late Effects of Radiation, 258.

Chapter 25. Phagocytosis

A. PHAGOCYTOSIS 7 261

1. Preparation of Phagocytic Cells, 262.Neutrophilic Polymorphonuclear Cells, 262: from Human Blood, 263, fromHorse Blood, 264, from Peritoneal Exudates, 264.Monocytes and Macrophages, 265. Sources: Peripheral Blood, 265; PeritonealExudates, 266; Phagocytes of the Unstimulated Peritoneal Cavity, 267; LungMacrophages: from Unstimulated Donors, 267, from Stimulated Donors, 268.Eosinophiles, 269: from Blood and Peritoneal Exudates, 269.

2. Media and Cell Counting, 270.

CONTENTS IX

Balanced-Salt Solutions, 270; Serum and Protein Additives, 271.Mononuclear Phagocytes in Tissue Culture, 271.Quantitation of Cell Mass, of Cell Counts, of Phagocytic Cells, 273.

3. Direct Evaluation of Ingestion, 273.Kinetics of Initial Rates of Phagocytosis, 273.Phagocytosis in Monolayers, 274; "Surface Phagocytosis," 274; Phagocytosisby Cells in Suspension, 275.Microscopy to Score Phagocytosis, 276; Electron Microscopy, 277.Inert Test Particles: Nonviable Bacteria, 277; Radiolabeled Bacteria andRadiolabeling Techniques, 278; Methyl-MC-Starch, 279; 125I-Protein Adsorbedto Bentonite, 279; Polystyrene Beads and Their Preparation, 280; Erythrocytes(Formalinized or Coated with Hemolysin), 282; Droplets of Paraffin Oil-OilRed 0, 283, and Lipopolysaccharide-Coated Oil Droplets, 284; Droplets ofDimethyl Phthalate, 284. Viable Organisms as Test Particles: BactericidalTesting, 285; Fungicidal Testing with Candida albicans, 288.

4. Evaluation of Phagocytosis by Measuring Phagocytic Metabolism, 288.Generation of Hydrogen Peroxide during Microbicidal Activity, 288; Oxida-tion of [1-14C]Glucose, 289; Reduction of Nitroblue Tetrazolium as a Measureof Rate of Ingestion, 291; Iodide-Protein Fixation by Cellular Myeloperoxi-dase, 292.

B. CLEARANCE in Vivo . 292

Principles, 292; Antigen-Antibody Complexes, 293, 295; Bacterial Clearance,293.

Clearance of Carbon from the Blood Stream, 294; Effect of RES Stimulation,295.Clearance of Bacteria from Serous Cavities, 295.

C. ELIMINATION OF S 1 CR-LABELED ERYTHROCYTES 296

Introduction, 296: Studies of Normal Erythrocyte Lifespan, 296; Assessmentof Anti-Erythrocyte Antibodies, 297; Patterns of Elimination, 297.Labeling and Sampling Methods, 298: Techniques, 298; Chromium Dose Cal-culation, 298; Blood Collections, 299.Elimination Rates, 299: Immediate Elimination by Natural Antibodies or byRES, 299; Immune Clearance, 299: Onset of Antibody Formation, Slow Im-mune Clearance, Calculations, 300; Sensitivity of the 5'Cr-Elimination Method,301.

Chapter 26. Antibody Synthesis in Vitro

A. TISSUE CULTURE METHODS FOR ANTIBODY PRODUCTION in Vitro . 303

1. Introduction, 303.2. Technique of Lymph Node Cultures, 303.

Immunization with T2 Bacteriophage, 303; Handling and Subdivision ofLymph Nodes, 304; Tissue Cultures, 304.

3. Culture of Spleen Cells for Primary Response, 305.

X CONTENTS

Preparation of Disrupted T2 Phage Antigen, 306; Dispersal of Spleen Cells,306; Culture of Cells with Phage Antigen, 306.

4. Measurement of Synthesized Antibody, 307.Phage. Neutralization Technique with Added Complement, 307; Incorporationof Radiolabeled Leucine or Lysine, 307; Kinetics of Antibody Synthesis, 308;

*." Concentration of Culture Fluids, 308. •• •

B. FEEDER CELL TECHNIQUES FOR ANTIBODY FORMATION In Vitro . 309

General Considerations, 309; Immunization by Diphtheria Toxoid in Rats,311, in Rabbits, 313. Stimulation of Immune Rat Lymph Node Fragments byDiffusible Materials Produced by Nurse .Cells-in a Parabiotic Chamber, 310;Stimulation of Immune Rabbit Lymph1 Node Fragments by Rabbit ThymusPreparations in Miniature Parabiotic Chamber, or by Thymosin, 313.

C. SINGLE-CELL TECHNIQUES I N ANTIBODY PRODUCTION . . . . 314

Introduction, 314.Micromanipulation Equipment, 315; Advance Preparation of the Cover Slipin the Micromanipulation Chamber, 318; Cell Depot Droplets for Selectionof Single Cells, 319, 321.Handling of Mammalian Cells, 318, 321: Isolation and Washing Procedures,321; Single Cell Isolation, 322; Special Bicarbonate-Free Nutrient Medium,323.Detection of Antibody to Bacterial Antigen Synthesized by Single Cells, 324:Bacterial Adherence to Single Cells, 324; Immobilization of Motile Bacteria,325; Observations on Bacterial H and 0 Agglutination, 325. Quantitation ofAntibody in Microdroplets by Serial Dilution, 326; Determination of Mer-captoethanol Sensitivity of Antibody, 326.Handling of Plaque-Forming Cells (PFC), 326: Liquid Monolayer Techniqueand Inversion of Micromanipulation System, 326; Carboxymethyl Cellulose

. • Plaque Technique, 327; Serial Transfers of PFC, 329.Micromanipulation and Autoradiography of Cells Labeled in Vitro, 330.Chromosomal Analysis of Antibody-Forming Cells Arrested in Metaphase,331: Deoxycholate Method of Rupturing Cells, 331; Microadaptations of theFord Technique, 332. . .Microradioiodination of PFC-Secretions, 333: Electrophoresis on CelluloseAcetate, 334; Autoradiography, 335; Microdensitometric Analysis, 335.

D. PLAQUE TECHNIQUES FOR RECOGNIZING INDIVIDUAL ANTIBODY-

FORMING CELLS 335

1. Standard Techniques, 337.Preparation of Cell Suspensions, 337; Plating and Plaque Counting, 339; Criti-cal Factors, 341; Spurious Plaques, 342; Background Plaque Counts, 343.

2. Indirect Techniques, 343.Plating Procedures, 344; Elimination of IgM Plaques, 345; Fluorescence Stain-ing, 347.

3. The Thin Layer Technique, 347.

CONTENTS XI

Ultrathin Agar Layers, 348; Autoradiography of Cells Incorporating TritiatedThymidine, 349.

4. Modified Techniques, 350.Microscope Slide Method, 350; The Two-Slide Sandwich for Duplicate Platingand Differential Development, 351.Carboxymethyl Cellulose as Supporting Medium, 352: Closed vs. Open Sys-tems, 353.Plaque Assays in Liquid Media, 353: Monolayers in Chambers, 354; Erythro-cyte Layer Bound to Petri Dish, 354; Discussion, 355.Microcultures, 356: Spot Tests for Hemolysin, 356; Microplaque Test, 356;Hemolytic Plaque Assays in Microculture Plates, 356.Plaque Assay for Antigen-Producing Cells with Hybrid Antibody, 357.

5. Hemolytic Plaques to Detect Synthesis of Antibodies to Diverse Antigens,357.General Conditions, 357: Density of Determinants, Fragility Testing, Inhibi-tion of Plaques by Free Antigen, 358.Cellular Responses to Protein Antigens, 359; Methods of Coupling, 359.Cellular Responses to Polysaccharide Antigens, 361.Anti-Hapten Antibodies, 362; Anti-Penicillin Antibody, 361.Plaque Technique to Detect Antibodies to Bacterial Surface Antigens, 364;Plating Technique for Bacteriolytic Plaques, 365.Plaque Technique with Lymphocytes as Target Cells, 365: Lymphoma H-2d

Target Cells, 365; Thymus Cells as Targets, 365; Special Procedures, 366;Titration of Cytolytic Antibodies, 366.

6. Plating Error and Efficiency of Plating, 366.Source of Variations in Plaque Numbers, 366; Preserved Standard Suspensionsof Plaque-Forming Cells as Reference, 367, 369.Analysis of Variance: the "Poisson Component" and Procedural Errors, 366.Plating Efficiency: Relative, 368, Absolute, 369.

E. ROSETTE-PLAQUE M E T H O D S FOR DETECTION OF ALLOTYPE AND

HAPTEN RECEPTORS I N PLAQUE-FORMING CELLS 370

Introduction, 370: Lymphocyte Receptor for Sheep Erythrocytes vs. DirectPlaque Technique, 370; Expression of Allot.ype Heterozygosity, 371.Detection of Individual Hapten Binding and Anti-Hapten Secreting Cells, 372:Preparation of Lymph Node Cells, Types of Target Cells, Rosette PlaqueAssay, 372.Rosette Formation vs. Plaquing: Lymphocytes from NlP-Immunized RabbitsPassively Sensitized by Anti-Rabbit Allotype Antibody, 373.

Chapter 27. Immunohistological Methods

A. PREPARATION OF T I S S U E FOR STUDY WITH LABELED ANTIBODY AND

LIGHT-MICROSCOPY 375

1. Introduction, 375: Preservation of Architecture of Tissue as Goal, 375; Theoryand Application, 377.

Xll CONTENTS

Sampling of Tissue, 377: Living Preparations, 378; Tissue Imprints ("Im-pression Smears") on Glass, 378; Tissue Blocks, 379.

2. Theory and ApplicationFast Freezing: Theory, 379; Use of Tissue Carriers, 381.Dehydration by Freeze-Substitution: Theory, 383; Practice, 407; DehydrationSolvents, 384; Temperature of Substitution, 385.Fixation: Theory 386; Direct Fixation, 387, 423; Post-Fixation, 389: of FrozenSections, 389, of Freeze-Substituted Tissue, 389; Secondary Fixation, 395.Embedding: Theory, 396; Ice and Aqueous Gums, 397; Diethylene GlycolDistearate, 399; Polyethylene Glycol Distearate and Carbowax, 400; Plastics,402.Sectioning, Theory, 402: Frozen Sections, 402, 420: Disadvantages, 404; Waxesand Paraffins, 405; Cutting of the Block, 406, Spreading of Sections, 406,Affixing to Slides, 407, Storage Conditions, 407.

3. Preparative MethodsThe Freeze-Substitution-Fixation Method, 407: Preparation of Freezing Bath,408; Preparation of the Substitution Bath, 410; Processing of Tissue, 412;Processing Tissue Culture Slides, 415.Examination Procedures, 416: Darkfield Inspection of Block Sections, 416;Phase Contrast Examination of Unhydrated Sections, 417; Empirical Choiceof Fixative for Special Problems, 417; Two-Step Test for Effectiveness ofFixation, 418; Testing with a Secondary Fixative for Completeness of Tissueand Antigen Insolubilizations, 419.Procedures when Antigen Is "Missing," 419.The Frozen-Section Method, 420; Test for Effectiveness of Fixation, 418.The Direct-Fixation Method, 423.

424B. FLUORESCENT ANTIBODY AS SPECIFIC CYTOCHEMICAL REAGENTS

1. Preparation of Fluorescein-Coupled Globulins, 425.Selection of Antiserum, and Assurance of Titer and Specificity, 425; Separationof Globulin, 425.Choice of Fluorochrome, 427; Conjugation Procedures, 429; Purification ofthe Conjugate, 429, and Fractionation on DEAE-Columns, 430; Analysis of theFluorescein: Protein Ratio, 433.Fluorescence Efficiency, 434: Comparison with Standard Sodium Fluorescein,434.Changes in Antibody Titer by Conjugation, 435; Testing for Specificity, 436.

2. Tissue Preparation for Immunofluorescence Staining7"437r~Quick Freezing, Cutting of Frozen Sections, 437; Preparing and HandlingParaffin Blocks, 438.

3. "Staining" with Labeled Antibody, 439.Direct Staining, 439; Indirect Staining ("Sandwich Technique"), 439.Staining of Complement in Tissue Sections, 440: Addition of Complement toUnknown Sera in Sandwich Technique, 441; Search for Complement Com-ponents in Tissues, 441.

4. Controls on Specificity of Fluorochrome Staining, 441.

CONTENTS Xlll

Inhibition of Specific Staining by Unlabeled Antibody, 442; Specific Removalof Labeled Antibody by Precipitation with Specific Antigen, 442; Failure toStain Control Tissues, 442; Exposure of Tissue to Labeled Antibody of OtherSpecificities, 443; Counterstains, 443.

5. Microscopic Equipment for Observing Fluorescence, 443.

6. Photography of Preparations Stained with Fluorescent Antibody, 443.

7. Tetramethylrhodamine Isothiocyanate (TRITC) Conjugates, 444.Properties of TRITC, 445; Conjugation with Purified Ig, 445; Purification ofConjugated Globulin, 446.

8. Visualization of Antigens by Fluorochrome Reagents of Contrasting Color,446.Staining and Inhibition Techniques, 446; The Ploem Incident Light Micro-scope for Examining TRITC-Fluorescence, 447.

9. Staining with FITC-Coupled and Ferritin-Coupled Univalent Antibody Frag-ments ("Hybrid Antibodies"), 447.Preparation of Fab' Fragments of Rabbit Anti-Vesicular Stomatitis Antibodyand of Rabbit Anti-Ferritin Antibody, 448; Reconstitution of Hybrid (FabOito Heterodimers, 448; Testing the Heterodimer for Attachment to Virus inMonolayers and to Ferritin and FITC-Coupled Apoferritin, 448.

C. FLUORESCENT PROTEINS FOR T H E DETERMINATION OF C E L L D E A T H 449

Principle, 449: Preparation of FITC-Labeled Bovine Albumin, 449; Detectionof Dead Cells: in Organ Cultures, 449, in Tissues in Vivo, 451.Fixation and Sectioning of Tissues, 451; Differentiation of Phagocytosed Fl-BSA and Uptake by Dead Cells, 451.

D. STAINING OF HISTONES IN NUCLEI OF LYMPHOID CELLS 451

Applicability: Lysine-Rich Histones in Smears, Frozen Cryostat Sections ofMammals, Aves, Plants, Squash Preparations, Electropherograms of IsolatedHistones on Cellulose Acetate, 452.Methods, 452: Formalin Fixation, Ammoniacal Silver, Formalin Developer,452; Cytophotometry for Relative Density of Stain, 455.Selective Staining of: Normal Lymphoid Cells vs. Antigen-Stimulated Animals,454, Nuclear Histones of Cancer Cells, 455.Comments: Noninterference by Prior or Subsequent Feulgen Staining, 453;Characterization of Cell Types by Altered Prestaining with Formalin, 456;Staining of Arginine-Rich Histones by Fast Green after TJN~A-Extraction, 456.

Chapter 28. Application of Electron Microscopy to Problems inImmunology

A. INTRODUCTION 457Study of Cells during Antibody Synthesis, Distribution of Antigens, Cell-Specific Antigenic Markers. Cytopathology, Antibody as Cytologic Stain, Re-quirement for Electron-Dense Antibody Molecules, 457.Complexing of Antibody with Ferritin, with Enzymes, with Viruses, 458.

XIV CONTENTS

B. I M M U N E LABELING FOR ELECTRON MICROSCOPY 458 r

1. Ferritin-Conjugated Antibody, 458.Introduction, 458: Surface Vs. Intracellular Staining, 459; Preliminary Trialswith Fluorescein-Ferritin Doubly Conjugated Antibody, 459.Surface Antigens on Cells, 459: Prefixation, 459.Intracellular Antigens, 460: Requirement for Limited Tissue Disruption, 461;Prefixation, 461; Methods for Tissue and Cell Disruption, 461.Ferritin-Antibody Staining, Post-Fixation and Embedment, 462.Controls on Specificity of Staining, 463: Use of Nonspecific Ferritin-GlobulinConjugate, 463, Other Considerations, 463.

2. Hybrid Antibody Markers for Electron Microscopy, 464.Principle: Proteins as Labels and Cellular Antigens as Target Joined via Bi-functional Hybrid Antibody, 464; Labels: Ferritin, Hemocyanin, Peroxidase,Viruses (SBMV, TYMV, TMV, VS), 464; Random Recombination of Fab'Fragments, 465: Initial Isolation of Specific Antibodies, 466.Purification of Some Labels: Ferritin, 466; Southern Bean Mosaic Virus, 468;Tobacco Mosaic Virus, 468.Purification of Immunoglobulins as Antigens, 469.Immunoadsorbents, 470: Preparation of Aminophenylbutyryl Aminoethyl(APB) Cellulose, Coupling with Antigen, 471; Coupling to Agarose Beads, 471.Immunization of Animals, 472; Recovery of IgG2 and IgGi, 473; Preparationof F(ab')2 Fragments, 473; Isolation of F(ab')2 on Immunoadsorbent, 473;Hybridization of F(ab')2 Antibodies via Borohydride Reduction to Fab', 474.Labeling of Cells: Directly with Hybrid Antibodies, 475, Indirectly with Pri-mary Antibody and Secondarily with Hybrid Antibody as "Sandwich," 476;Labeling of Cells in Suspension, 476.Examples and Critique, 477.

3. Enzyme-Labeled Antibody Markers for Electron Microscopy, 482.Principle: Local Accumulation of Catalytic Products, 482; PreembeddingStaining for Surface Localization vs. Postembedding Staining for DiffusionLocalization, 483.Fixation, 483: for Diffusion Localization, 484, for Surface Localization, 484.Preparation of Tissues to Localize Intracellular Antigen, 485. Testing theFixative for Loss of Antigenicity, 486.Preparation of Antibodies and Fragments, 486: Purification of MonovalentPapain-Antibody Fragments by Affinity Chromatography for Diffusion Lo-calization, 487, of Bivalent Pepsin-Antibody Fragments for Surface Localiza-tion, 488.Enzyme Labels: Horseradish Peroxidase, Cytochrome c, 11-Microperoxidase,8-Microperoxidase, 489.Choice of Conjugation Reagents, 489: Random Polymerizations vs. Two-StepProcedures, 489; Preparation of iV-Hydroxysuccinamide Ester of p-Formyl-benzoic Acid, 490.Coupling Procedures, 490: Horseradish Peroxidase- Fab Fragments, 490; 8-Microperoxidase-Fab Fragments, 491.

CONTENTS XV

Practical Applications in Localizing Antigens, 492: Diffusion Localization byIndirect Sandwich Technique, 492, 493; Surface Localization on Thin Sectionsby Sandwich Technique, 493.Controls: Immunological, 495; Determination of Endogenous PeroxidaticActivity of Tissue and Its Inhibition, 495; Test for Permeation of Reagentsinto Cells, 495.

C. ELECTRON MICROSCOPY OF IMMUNOGLOBULINS AND I M M U N E

COMPLEXES 495

General Considerations for Visualizing Soluble Proteins by Negative Contrast,495, 498.Support Films: Carbon Covered Nitrocellulose Layers or Adhesive-Glue Grids,497.

Negative-Staining Heavy-Metal Salts, 498.Properties of Protein Molecules Capable of Visualization, 499.Staining Procedures, 499: Sequential Drop Method Allowing Reactions onGrids before Staining, 500; One-Step Direct Staining, 500; Carbon FilmTransfer Method, from Mica, 501..Immunological Applications, 502: Rabbit IgG Linked by Bivalent Hapten forStructures of IgG, Fc, Fab, 502; Complement Fixation by Antigen-AntibodyTetramers, 503; Human IgA Dimer, 503, 504; IgM Ultrastructure, 505; ClqUltrastructure, 506; C3 Component of Complement, 506. .

AUTHOR INDEX 507

SUBJECT INDEX 523