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Part Ⅰ．Theory and Practice of Histological Technic:General Applications1

Chapter 1 Histological Specimens3

Sources and Types of Material4

Procurement of Specimens5

Normal Tissues5

Pathological Tissues8

Embryos and Cultured Tissue8

Kinds of Preparations9

Whole Mounts10

Sections11

Teased Preparations11

Smears12

Living and Preserved Material15

Labels17

Chapter 2 Some Phenomena Related to Tissues19

The Living and the Dead19

Cellular Membranes in Unfixed and Fixed Tissues21

Osmotic Pressure21

Isotonic,Hypertonic and Hypotonic Solutions22

Source of Osmotic Pressure22

Calculation of an Isotonic Solution23

Isotonic Solutions Containing a Mixture of Ingredients23

Hydrogen Ion Concentration24

The Meaning of pH24

Strength of Acids and Bases25

Buffer Solutions27

Chapter 3 Fixation29

Effects of Fixing Agents29

Properties of Fixing Fluids30

Solubilities and Solutions of Reagents33

Effects of Osmotic Pressure35

Classification and Composition of Fixing Fluids35

Choice of Fixation38

General Comments on Fixation41

Chapter 4 Washing,Dehydrating and Clearing43

Washing43

Wetting46

Dehydration48

Technics of Dehydration50

Clearing52

Chapter 5 Embedding53

Special Properties of Embedding Masses53

Choice of Embedding Medium54

1．Water-Soluble Masses55

A．Gelatin and Gums55

B．Polyethylene Glycol55

2．Water-Insoluble Masses56

A．Paraffin56

B．Nitrocellulose57

Sumary of Factors Determining the Type of Embedding58

Embedding in Water-Soluble Masses58

1．Gelatin58

2．Masses Derived from Plant Sources59

3．Polyethylene Glycol66

Embedding in Paraffin60

Grades and Properties61

Ovens62

Filtration of Paraffin63

Permeation of Specimens63

Casting65

Modified Paraffin Embedding Masses68

Embedding in Nitrocellulose69

Names and Properties of Materials69

Embedding Solutions71

Permeation of Specimens73

Gelation of Nitrocellulose Solutions75

Technics of Embedding with Nitrocellulose76

Miscellaneous Comments and Cautions79

Double Embedding80

Embeddingin Plastic81

Methacrylate Embedding82

Chapter 6 Equipment for Sectioning:Knife Sharpening83

Technics83

Sectioning with a Razor83

Sectioning with a Regular Microtome84

Microtome Knives and Accessories87

Blades87

Backs87

Handles88

Safety Razor Blade Holders88

Knife Sharpening89

Edge of the Knife90

Grinding and Polishing90

1．Hones91

2．Technic of Honing91

3．Honing with a Lap of Plate Glass93

4．Stropping94

Honing Machines98

Requirements for Ultrathin Sectioning99

Glass Knives100

Steel Knives100

Chapter 7 Technic of Sectioning105

Frozen Sections105

Technic106

Paraffin Sections108

Affixing Blocks to Carriers108

Sectioning109

Formation of Ribbons112

Handling and Storing114

Nitrocellulose Sections115

Block Carriers116

Mounting of Blocks116

Technic of Cutting117

Serial Sections118

Disposition of Waste Embedding Material120

Care of Block Carriers120

Sectioning Doubly Embedded Material120

Chapter 8 Mounting and Covering121

Mounting Paraffin Sections121

Coating Slides with Albumen Adhesive122

Affixing Sections to Slide123

Practical Suggestions125

Covering Mounted Paraffin Sections127

Mounting and Covering Nitrocellulose Sections127

Attaching Unstained Sections127

Mounting Stained Sections128

Flattening Covered Nitrocellulose Sections130

Mounting and Covering Frozen Sections131

Mounting Media132

Water-Soluble Mounting Media132

Water-Insoluble Mounting Media133

Chapter 9 Staining135

Types of Staining Agents136

Synthetic Dyes136

Water-Soluble Dye Reactions137

Water-Insoluble Dye Reactions137

Natural Dyes138

Metallic Stains139

Pigments140

General Procedure of Staining140

Glassware,Hydration and Dehydration141

Paraffin Sections141

Nitrocellulose Sections144

Frozen Sections144

Progressive and Regressive Staining145

Differentiation146

Mordants149

Dehydrating,Clearing and Mounting150

Dehydration150

Clearing152

Mounting Media152

Cover Glasses154

Shape,Size and Thickness154

Cleaning Cover Glasses155

Staining Technics156

Original Methods and Modifications156

The Steps of a Technic157

Abridgment in Descriptions of Technics157

Stains and Chemicals158

Stains158

Chemicals159

Solutions Based on Percentage159

Solutions Based on Normality161

Part Ⅱ．Formulas and Specific Application of Procedures163

Chapter 10 Fixing Fluids165

F1．Formalin solution165

F2．Formalin-acetic165

F3．Formalin-picric-acetic166

F3a．Formalin-picric-trichloracetic166

F4．Carnoy＇s fluids166

F5．Carnoy-Lebrun fluid167

F6．Ohlmacher＇s fluid167

F7．Zenker＇s fluid167

F8．Müller＇s fluid168

F9．Orth＇s fluid168

F9a．Regaud＇s fluid168

F10．Sublimate-acetic168

F11．Zenker-formalin mixtures168

F12．Gilson＇s fluid169

F13．Petrunkewitch＇s nitric-acetic-sublimate169

F14w,F14s．Flemming＇s fluids169

F15．Flemming＇s fluid without acetic acid170

F16．Chamberlain＇s fixative170

F17．Navashin＇s fluid171

F18．Randolph＇s CRAF171

F19．Champy＇s fluid171

F20．Heidenhain＇s SUSA171

F21-1,F21-2．Worcester＇s fluids172

F22．Sublimate-formalin-acetic(for plant tissues)172

F23．Variation of formalin-mercuric chloride fixative173

Decalcifying Fluids173

F24．Alcoholic nitric acid for decalcification173

F25．Formic acid-sodium citrate mixture173

F26．Chelating agent for decalcification174

Chapter 11 Staining Solutions175

Hematoxylin Solutions175

S1．Weigert-type stain176

S2．Mayer＇s hemalum178

S3．Mayer＇s acidified hematoxylin178

S4．Ehrlich＇s acid hematoxylin178

S5．Harris＇formula179

S6．Delafield＇s hematoxylin solution179

S7．Mallory＇s phosphotungstic acid hematoxylin179

S8．Heidenhain method180

Carmine Solutions181

S9．Grenacher＇s alum-carmine solution181

S10．Grenacher＇s borax carmine181

S11．Mayer＇s carmalum182

S12．Mayer＇s paracarmine182

S13．Acetocarmine182

S14．Moree＇s iron-acetocarmine mixtures183

Solutions of Synthetic Dyes and Other Staining Agents184

S15．Aldehyde-fuchsin184

S16．Schiff＇s leucofuchsin reagent186

Chapter 12 Staining in the Block189

Block Staining with Dyes189

One-Color Block Stains189

M1．Carmine189

M2．Hematoxylin192

M3．Toluidine blue method193

Two-Color Block Stain194

M4．Picro-Feulgen method194

Block Staining by Metallic Impregnation195

1．Osmic Acid195

M5．Bruesch＇s(1942)procedure195

M6．Marchi＇s method,Swank-Davenport modification196

M7．Myelin-sheath stain forperipheral nerves198

M8．Kopsch＇s method for the Golgi apparatus198

2．Gold Chloride199

M9．Ranvier＇s(1880)method199

M10．Wunderer＇s(1908)method200

M11．Method of Corrington201

3．Silver Nitrate202

M12．Demonstration of intercellular boundaries202

Stains for Nervous Tissue203

A．Golgi-Type Stain203

M13．Golgi＇s original method204

M14．Golgi＇s rapid process205

M15．Porter＇s and Davenport＇s(1949)modification of the Golgi method206

M16．Fox＇s(1951) zinc chromate-Colgi method206

B．Cajal-Type Stain207

M17．Cajal＇s formula 3208

M18．Ranson＇s(1911)pyridine-silver method209

M19．Perez(1931)-Nonidez(1939)method210

C．Bielschowsky-Type Stain211

M20．Bielschowsky＇s(1909)method,slightly modified212

4．Mercuric Chloride214

M21．Golgi-Cox method214

Chapter 13 Staining Sections217

Section Stains with Dyes217

Single-Dye Stains217

1．Iron-Hematoxylin Methods217

A．For Mitochondria218

M22．Regaud＇s method218

B．For Nuclei219

M23．Iron-hematoxylin differentiated by picric acid219

M24．Mordanting fixation for iron-hematoxylin219

C．For Myelin Sheaths220

M25．Pal-Weigert method;Kapper＇s modification220

M26．Pal-Weigert method;Clark and Ward(1943)modification221

2．A Stain for General Use222

M27．Chlorazol black E222

3．Stains for Nerve Cells223

M28．Thionin stains223

A．Progressive Staining223

B．Regressive Staining224

M29．Cresyl violet stain224

M30．Cresyl violet acetate stain for frozen sections225

4．Oil-Soluble Dyes for Staining Lipids225

M31．Oil-soluble dye in a water-alcohol mixture226

Double-Dye Stains227

1．Hematoxylin-Eosin Methods227

M32．Delafield＇s hematoxylin and eosin Y228

M33．Hematoxylin-eosin stain for formalin-fixed tissue229

2．Azures and Eosins231

M34．Wright＇s method for blood232

M35．Giemsa stain233

M36．Lillie＇s azure A-eosin B method for sections234

M37．Haynes＇ azure-eosin stain for tissue235

M38．Mallory＇s phloxine-methylene blue stain235

M39．Field＇s method for blood parasites236

3．Safranin and Fast Green237

M40a．Safranin-fast green method237

M40b．Safranin-fast green method237

4．Gram＇s Stain238

M41．Gram stain for bacteria in tissues238

M42．Hucker＇s modification of the gram stain for bacteria239

5．Methyl Green and Pyronin(Basic Dyes)240

M43．Methyl green-pyronin method240

Multiple-Dye Stains243

M44．Van Gieson＇s stain(with hematoxylin)244

M45．Massonn＇s trichrome stain(1928),supplemented from Foot(1933)245

M46．Aniline blue collagen stain246

M47．Pianese Ⅲb stain(1896)247

M48．Flemming＇s triple stain(1891),as modified for plant material by Margolena(1935)247

M49．Quad stain248

Section Stains with Metals249

Silver Impregnations250

1．Cajal Type250

M50．Staining nerve fibers in unmounted nitrocellulose sections250

2．Bielschowsky Type251

M51．Staining peripheral nerve elements252

M52．Roger＇s method for paraffin sections253

M53．Gold toning254

3．Direct Impregnation with Diammino Silver255

Neurological Methods255

M54．Del Rio Hortega＇s method for oligodendroglia256

M55．Staining neuroglia in paraffin sections257

M56．Staining oligodendroglia and microglia in nitrocellulose sections257

Methods for Connective Tissue257

M57．Periodic acid-Foot stain for connective tissue258

4．Silver Proteinate Staining(Bodian Type)259

M58．Bodian＇s Protargol method259

M59．Two-hour silver method261

Impregnation with Gold261

M60．Cajal＇s gold-sublimate method261

Part Ⅲ．Histochemistry263

Chapter 14 Historical Introduction;Objects and Requirements of Histochemistry265

Introduction and Objectives265

Requirements for Histochemical Technics267

Chapter 15 Inorganic Constituents of Tissues271

Anionic Material271

Chlorides271

M61．Method for chlorides271

Fluoride,Bromide and Iodide272

Carbonate273

Sulfate274

M62．Method for free sulfate274

Phosphate275

M63．Phosphate in nucleic acids275

Other anions276

Cationic Material276

Sodium276

Potassium277

M64．Method for potassium277

Calcium and Magnesium278

M65．Dahl＇s method for calcium279

Iron and Copper280

M66．The ferrocyanide＇reaction281

M67．The demonstration of copper by dithiooxamide283

Masked Iron283

M68．Nonionized iron283

M69．Schmelzer＇s thiocyanate method for iron283

Trace Elements284

Chapter 16 Organic Constituents of Tissues:Polysaccharides287

Group Ⅰ．Carbohydrate Type288

Starch289

M70．Durable iodine stain for cellulose,starch and glycogen290

Glycogen290

M71．Best＇s carmine stain for glycogen294

Fructans(Fructosans)295

M72．Tests for inulin296

Galactan(Galactogen)296

Cellulose297

M73．Iodine-lithium chloride method for cellulose297

Group Ⅱ．Mucopolysaccharides298

Neutral Mucopolysaccharides299

Chitin299

Pectin300

M74．Ruthenium red stains301

Hemicellulose302

Acid Mucopolysaccharides303

Simple Acid Mucopolysaccharides303

Complex Acid Mucopolysaccharides304

M75．Metachromatic staining;method of Kramer and Windrum305

Basophilia306

Iron Absorption by Acid Mucopolysaccharides307

M76．Hale＇s method for acid polysaccharides in animal tissues307

M77．Ferric mannitol(pH5．0)method of Lillie and Mowry308

Periodic Acid-Schiff (PAS)Procedure for Polysaccharides309

Chapter 17 Organic Constituents of Tissues:Proteins and Amino Acids311

Proteins311

The Gram Reaction313

M78．Gram＇s stain for tissue314

Critical Analysis of Protein Staining315

Demonstration of Protein in General316

M79．Mercuric-bromphenol blue staining of protein316

Amino Acids317

M80．Protein-bound amino radicals318

M81．The ninhydrin reaction319

Tyrosine320

M82．Millon＇s reaction for tyrosine320

M83．Histochemical recognition of phenols321

Histidine,Tryptophane and Tyrosine322

Tetrazotized Benzidine322

M84．Azo reaction with coupling323

Tryptophane323

M85．The tryptophane reaction323

M86．Reaction for 3-indolyl derivatives324

M87．Method for derivatives of indole324

Arginine325

M88．The Sakaguchi reaction for arginine325

M89．The improved Sakaguchi reaction adapted to histochemistry326

Cysteine,Cystine and Methionine327

M90．Nitroprusside test for SH radicals328

M91．Ferric ferricyanide reduction329

M92．Protein-bound sulfhydryl330

M93．Demonstration of sulfhydryl and disulfide331

M94．Demonstration of disulfide331

Chapter 18 Organic Constituents of Tissues:Lipids333

Classification of Typical Lipids333

Atypical Lipids and Lipoproteins335

Means of Identification of Lipids337

Fats and Phospholipids338

M95．Neutral fat and fatty acid338

M96．Baker＇s test for phospholipids339

M97．The plasmal reaction340

Sterols and Steryl Esters342

M98．Acetic-sulfuric test for cholesterol;original version from Schultz342

M99．Bismuth trichloride method for sterols343

Waxes344

Rubber345

M100．Oil blue NA stain for latex345

M101．Frozen-section method for latex345

Chapter 19 Enzymes347

Hydrolases349

Esterases349

M102．Naphthyl acetate method for esterases351

M103．The Tween method for lipase-type esterases351

M104．Demonstration of esterase by the production of an indigo dye352

Acid and Alkaline Phosphatases353

M105．Alkaline phosphatase;cobalt sulfide method353

M106．A1kaline phosphatase by an azo-dye reaction354

M107．Acid phosphatase;dye method355

M108．Lead acetate method for acid phosphatase356

M109．Phosphamidase357

Other Hydrolases358

Transferases and Oxidoreductases358

Oxidases360

M110．Dihydroxyphenylalanine oxidase reaction361

M111．Demonstration of tryosinase,as used by Fitzpatrick,et al．and by Foster and Cook361

M112．Indophenol oxidase(cytochrome oxidase)reactions362

Peroxidases364

M113．The benzidine method for blood pigments365

M114．The Lison-Dunn leuco-dye method for peroxidase366

Dehydrogenases67

M115．Succinic dehydrogenase localization368

Lyases and Syntheases368

M116．Demonstration of carbonic anhydrase369

Isomerases and Racemases370

Chapter 20 Applications of Schiff＇s Reagent371

M117．Nucleal reaction of Feulgen and Rossenbeck372

M118．Periodic acid-Schiff(PAS)reaction373

M119．Peracetic acid-Schiff reaction374

Concluding Comments on Histochemical Methods375

Bibliography377

Index389