Histological and Histochemical TechnicsPDF电子书下载

Part Ⅰ．Theory and Practice of Histological Technic:General Applications 1

Chapter 1 Histological Specimens 3

Sources and Types of Material 4

Procurement of Specimens 5

Normal Tissues 5

Pathological Tissues 8

Embryos and Cultured Tissue 8

Kinds of Preparations 9

Whole Mounts 10

Sections 11

Teased Preparations 11

Smears 12

Living and Preserved Material 15

Labels 17

Chapter 2 Some Phenomena Related to Tissues 19

The Living and the Dead 19

Cellular Membranes in Unfixed and Fixed Tissues 21

Osmotic Pressure 21

Isotonic,Hypertonic and Hypotonic Solutions 22

Source of Osmotic Pressure 22

Calculation of an Isotonic Solution 23

Isotonic Solutions Containing a Mixture of Ingredients 23

Hydrogen Ion Concentration 24

The Meaning of pH 24

Strength of Acids and Bases 25

Buffer Solutions 27

Chapter 3 Fixation 29

Effects of Fixing Agents 29

Properties of Fixing Fluids 30

Solubilities and Solutions of Reagents 33

Effects of Osmotic Pressure 35

Classification and Composition of Fixing Fluids 35

Choice of Fixation 38

General Comments on Fixation 41

Chapter 4 Washing,Dehydrating and Clearing 43

Washing 43

Wetting 46

Dehydration 48

Technics of Dehydration 50

Clearing 52

Chapter 5 Embedding 53

Special Properties of Embedding Masses 53

Choice of Embedding Medium 54

1．Water-Soluble Masses 55

A．Gelatin and Gums 55

B．Polyethylene Glycol 55

2．Water-Insoluble Masses 56

A．Paraffin 56

B．Nitrocellulose 57

Sumary of Factors Determining the Type of Embedding 58

Embedding in Water-Soluble Masses 58

1．Gelatin 58

2．Masses Derived from Plant Sources 59

3．Polyethylene Glycol 66

Embedding in Paraffin 60

Grades and Properties 61

Ovens 62

Filtration of Paraffin 63

Permeation of Specimens 63

Casting 65

Modified Paraffin Embedding Masses 68

Embedding in Nitrocellulose 69

Names and Properties of Materials 69

Embedding Solutions 71

Permeation of Specimens 73

Gelation of Nitrocellulose Solutions 75

Technics of Embedding with Nitrocellulose 76

Miscellaneous Comments and Cautions 79

Double Embedding 80

Embeddingin Plastic 81

Methacrylate Embedding 82

Chapter 6 Equipment for Sectioning:Knife Sharpening 83

Technics 83

Sectioning with a Razor 83

Sectioning with a Regular Microtome 84

Microtome Knives and Accessories 87

Blades 87

Backs 87

Handles 88

Safety Razor Blade Holders 88

Knife Sharpening 89

Edge of the Knife 90

Grinding and Polishing 90

1．Hones 91

2．Technic of Honing 91

3．Honing with a Lap of Plate Glass 93

4．Stropping 94

Honing Machines 98

Requirements for Ultrathin Sectioning 99

Glass Knives 100

Steel Knives 100

Chapter 7 Technic of Sectioning 105

Frozen Sections 105

Technic 106

Paraffin Sections 108

Affixing Blocks to Carriers 108

Sectioning 109

Formation of Ribbons 112

Handling and Storing 114

Nitrocellulose Sections 115

Block Carriers 116

Mounting of Blocks 116

Technic of Cutting 117

Serial Sections 118

Disposition of Waste Embedding Material 120

Care of Block Carriers 120

Sectioning Doubly Embedded Material 120

Chapter 8 Mounting and Covering 121

Mounting Paraffin Sections 121

Coating Slides with Albumen Adhesive 122

Affixing Sections to Slide 123

Practical Suggestions 125

Covering Mounted Paraffin Sections 127

Mounting and Covering Nitrocellulose Sections 127

Attaching Unstained Sections 127

Mounting Stained Sections 128

Flattening Covered Nitrocellulose Sections 130

Mounting and Covering Frozen Sections 131

Mounting Media 132

Water-Soluble Mounting Media 132

Water-Insoluble Mounting Media 133

Chapter 9 Staining 135

Types of Staining Agents 136

Synthetic Dyes 136

Water-Soluble Dye Reactions 137

Water-Insoluble Dye Reactions 137

Natural Dyes 138

Metallic Stains 139

Pigments 140

General Procedure of Staining 140

Glassware,Hydration and Dehydration 141

Paraffin Sections 141

Nitrocellulose Sections 144

Frozen Sections 144

Progressive and Regressive Staining 145

Differentiation 146

Mordants 149

Dehydrating,Clearing and Mounting 150

Dehydration 150

Clearing 152

Mounting Media 152

Cover Glasses 154

Shape,Size and Thickness 154

Cleaning Cover Glasses 155

Staining Technics 156

Original Methods and Modifications 156

The Steps of a Technic 157

Abridgment in Descriptions of Technics 157

Stains and Chemicals 158

Stains 158

Chemicals 159

Solutions Based on Percentage 159

Solutions Based on Normality 161

Part Ⅱ．Formulas and Specific Application of Procedures 163

Chapter 10 Fixing Fluids 165

F1．Formalin solution 165

F2．Formalin-acetic 165

F3．Formalin-picric-acetic 166

F3a．Formalin-picric-trichloracetic 166

F4．Carnoy＇s fluids 166

F5．Carnoy-Lebrun fluid 167

F6．Ohlmacher＇s fluid 167

F7．Zenker＇s fluid 167

F8．Müller＇s fluid 168

F9．Orth＇s fluid 168

F9a．Regaud＇s fluid 168

F10．Sublimate-acetic 168

F11．Zenker-formalin mixtures 168

F12．Gilson＇s fluid 169

F13．Petrunkewitch＇s nitric-acetic-sublimate 169

F14w,F14s．Flemming＇s fluids 169

F15．Flemming＇s fluid without acetic acid 170

F16．Chamberlain＇s fixative 170

F17．Navashin＇s fluid 171

F18．Randolph＇s CRAF 171

F19．Champy＇s fluid 171

F20．Heidenhain＇s SUSA 171

F21-1,F21-2．Worcester＇s fluids 172

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

F23．Variation of formalin-mercuric chloride fixative 173

Decalcifying Fluids 173

F24．Alcoholic nitric acid for decalcification 173

F25．Formic acid-sodium citrate mixture 173

F26．Chelating agent for decalcification 174

Chapter 11 Staining Solutions 175

Hematoxylin Solutions 175

S1．Weigert-type stain 176

S2．Mayer＇s hemalum 178

S3．Mayer＇s acidified hematoxylin 178

S4．Ehrlich＇s acid hematoxylin 178

S5．Harris＇formula 179

S6．Delafield＇s hematoxylin solution 179

S7．Mallory＇s phosphotungstic acid hematoxylin 179

S8．Heidenhain method 180

Carmine Solutions 181

S9．Grenacher＇s alum-carmine solution 181

S10．Grenacher＇s borax carmine 181

S11．Mayer＇s carmalum 182

S12．Mayer＇s paracarmine 182

S13．Acetocarmine 182

S14．Moree＇s iron-acetocarmine mixtures 183

Solutions of Synthetic Dyes and Other Staining Agents 184

S15．Aldehyde-fuchsin 184

S16．Schiff＇s leucofuchsin reagent 186

Chapter 12 Staining in the Block 189

Block Staining with Dyes 189

One-Color Block Stains 189

M1．Carmine 189

M2．Hematoxylin 192

M3．Toluidine blue method 193

Two-Color Block Stain 194

M4．Picro-Feulgen method 194

Block Staining by Metallic Impregnation 195

1．Osmic Acid 195

M5．Bruesch＇s(1942)procedure 195

M6．Marchi＇s method,Swank-Davenport modification 196

M7．Myelin-sheath stain forperipheral nerves 198

M8．Kopsch＇s method for the Golgi apparatus 198

2．Gold Chloride 199

M9．Ranvier＇s(1880)method 199

M10．Wunderer＇s(1908)method 200

M11．Method of Corrington 201

3．Silver Nitrate 202

M12．Demonstration of intercellular boundaries 202

Stains for Nervous Tissue 203

A．Golgi-Type Stain 203

M13．Golgi＇s original method 204

M14．Golgi＇s rapid process 205

M15．Porter＇s and Davenport＇s(1949)modification of the Golgi method 206

M16．Fox＇s(1951) zinc chromate-Colgi method 206

B．Cajal-Type Stain 207

M17．Cajal＇s formula 3 208

M18．Ranson＇s(1911)pyridine-silver method 209

M19．Perez(1931)-Nonidez(1939)method 210

C．Bielschowsky-Type Stain 211

M20．Bielschowsky＇s(1909)method,slightly modified 212

4．Mercuric Chloride 214

M21．Golgi-Cox method 214

Chapter 13 Staining Sections 217

Section Stains with Dyes 217

Single-Dye Stains 217

1．Iron-Hematoxylin Methods 217

A．For Mitochondria 218

M22．Regaud＇s method 218

B．For Nuclei 219

M23．Iron-hematoxylin differentiated by picric acid 219

M24．Mordanting fixation for iron-hematoxylin 219

C．For Myelin Sheaths 220

M25．Pal-Weigert method;Kapper＇s modification 220

M26．Pal-Weigert method;Clark and Ward(1943)modification 221

2．A Stain for General Use 222

M27．Chlorazol black E 222

3．Stains for Nerve Cells 223

M28．Thionin stains 223

A．Progressive Staining 223

B．Regressive Staining 224

M29．Cresyl violet stain 224

M30．Cresyl violet acetate stain for frozen sections 225

4．Oil-Soluble Dyes for Staining Lipids 225

M31．Oil-soluble dye in a water-alcohol mixture 226

Double-Dye Stains 227

1．Hematoxylin-Eosin Methods 227

M32．Delafield＇s hematoxylin and eosin Y 228

M33．Hematoxylin-eosin stain for formalin-fixed tissue 229

2．Azures and Eosins 231

M34．Wright＇s method for blood 232

M35．Giemsa stain 233

M36．Lillie＇s azure A-eosin B method for sections 234

M37．Haynes＇ azure-eosin stain for tissue 235

M38．Mallory＇s phloxine-methylene blue stain 235

M39．Field＇s method for blood parasites 236

3．Safranin and Fast Green 237

M40a．Safranin-fast green method 237

M40b．Safranin-fast green method 237

4．Gram＇s Stain 238

M41．Gram stain for bacteria in tissues 238

M42．Hucker＇s modification of the gram stain for bacteria 239

5．Methyl Green and Pyronin(Basic Dyes) 240

M43．Methyl green-pyronin method 240

Multiple-Dye Stains 243

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

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

M46．Aniline blue collagen stain 246

M47．Pianese Ⅲb stain(1896) 247

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

M49．Quad stain 248

Section Stains with Metals 249

Silver Impregnations 250

1．Cajal Type 250

M50．Staining nerve fibers in unmounted nitrocellulose sections 250

2．Bielschowsky Type 251

M51．Staining peripheral nerve elements 252

M52．Roger＇s method for paraffin sections 253

M53．Gold toning 254

3．Direct Impregnation with Diammino Silver 255

Neurological Methods 255

M54．Del Rio Hortega＇s method for oligodendroglia 256

M55．Staining neuroglia in paraffin sections 257

M56．Staining oligodendroglia and microglia in nitrocellulose sections 257

Methods for Connective Tissue 257

M57．Periodic acid-Foot stain for connective tissue 258

4．Silver Proteinate Staining(Bodian Type) 259

M58．Bodian＇s Protargol method 259

M59．Two-hour silver method 261

Impregnation with Gold 261

M60．Cajal＇s gold-sublimate method 261

Part Ⅲ．Histochemistry 263

Chapter 14 Historical Introduction;Objects and Requirements of Histochemistry 265

Introduction and Objectives 265

Requirements for Histochemical Technics 267

Chapter 15 Inorganic Constituents of Tissues 271

Anionic Material 271

Chlorides 271

M61．Method for chlorides 271

Fluoride,Bromide and Iodide 272

Carbonate 273

Sulfate 274

M62．Method for free sulfate 274

Phosphate 275

M63．Phosphate in nucleic acids 275

Other anions 276

Cationic Material 276

Sodium 276

Potassium 277

M64．Method for potassium 277

Calcium and Magnesium 278

M65．Dahl＇s method for calcium 279

Iron and Copper 280

M66．The ferrocyanide＇reaction 281

M67．The demonstration of copper by dithiooxamide 283

Masked Iron 283

M68．Nonionized iron 283

M69．Schmelzer＇s thiocyanate method for iron 283

Trace Elements 284

Chapter 16 Organic Constituents of Tissues:Polysaccharides 287

Group Ⅰ．Carbohydrate Type 288

Starch 289

M70．Durable iodine stain for cellulose,starch and glycogen 290

Glycogen 290

M71．Best＇s carmine stain for glycogen 294

Fructans(Fructosans) 295

M72．Tests for inulin 296

Galactan(Galactogen) 296

Cellulose 297

M73．Iodine-lithium chloride method for cellulose 297

Group Ⅱ．Mucopolysaccharides 298

Neutral Mucopolysaccharides 299

Chitin 299

Pectin 300

M74．Ruthenium red stains 301

Hemicellulose 302

Acid Mucopolysaccharides 303

Simple Acid Mucopolysaccharides 303

Complex Acid Mucopolysaccharides 304

M75．Metachromatic staining;method of Kramer and Windrum 305

Basophilia 306

Iron Absorption by Acid Mucopolysaccharides 307

M76．Hale＇s method for acid polysaccharides in animal tissues 307

M77．Ferric mannitol(pH5．0)method of Lillie and Mowry 308

Periodic Acid-Schiff (PAS)Procedure for Polysaccharides 309

Chapter 17 Organic Constituents of Tissues:Proteins and Amino Acids 311

Proteins 311

The Gram Reaction 313

M78．Gram＇s stain for tissue 314

Critical Analysis of Protein Staining 315

Demonstration of Protein in General 316

M79．Mercuric-bromphenol blue staining of protein 316

Amino Acids 317

M80．Protein-bound amino radicals 318

M81．The ninhydrin reaction 319

Tyrosine 320

M82．Millon＇s reaction for tyrosine 320

M83．Histochemical recognition of phenols 321

Histidine,Tryptophane and Tyrosine 322

Tetrazotized Benzidine 322

M84．Azo reaction with coupling 323

Tryptophane 323

M85．The tryptophane reaction 323

M86．Reaction for 3-indolyl derivatives 324

M87．Method for derivatives of indole 324

Arginine 325

M88．The Sakaguchi reaction for arginine 325

M89．The improved Sakaguchi reaction adapted to histochemistry 326

Cysteine,Cystine and Methionine 327

M90．Nitroprusside test for SH radicals 328

M91．Ferric ferricyanide reduction 329

M92．Protein-bound sulfhydryl 330

M93．Demonstration of sulfhydryl and disulfide 331

M94．Demonstration of disulfide 331

Chapter 18 Organic Constituents of Tissues:Lipids 333

Classification of Typical Lipids 333

Atypical Lipids and Lipoproteins 335

Means of Identification of Lipids 337

Fats and Phospholipids 338

M95．Neutral fat and fatty acid 338

M96．Baker＇s test for phospholipids 339

M97．The plasmal reaction 340

Sterols and Steryl Esters 342

M98．Acetic-sulfuric test for cholesterol;original version from Schultz 342

M99．Bismuth trichloride method for sterols 343

Waxes 344

Rubber 345

M100．Oil blue NA stain for latex 345

M101．Frozen-section method for latex 345

Chapter 19 Enzymes 347

Hydrolases 349

Esterases 349

M102．Naphthyl acetate method for esterases 351

M103．The Tween method for lipase-type esterases 351

M104．Demonstration of esterase by the production of an indigo dye 352

Acid and Alkaline Phosphatases 353

M105．Alkaline phosphatase;cobalt sulfide method 353

M106．A1kaline phosphatase by an azo-dye reaction 354

M107．Acid phosphatase;dye method 355

M108．Lead acetate method for acid phosphatase 356

M109．Phosphamidase 357

Other Hydrolases 358

Transferases and Oxidoreductases 358

Oxidases 360

M110．Dihydroxyphenylalanine oxidase reaction 361

M111．Demonstration of tryosinase,as used by Fitzpatrick,et al．and by Foster and Cook 361

M112．Indophenol oxidase(cytochrome oxidase)reactions 362

Peroxidases 364

M113．The benzidine method for blood pigments 365

M114．The Lison-Dunn leuco-dye method for peroxidase 366

Dehydrogenases 67

M115．Succinic dehydrogenase localization 368

Lyases and Syntheases 368

M116．Demonstration of carbonic anhydrase 369

Isomerases and Racemases 370

Chapter 20 Applications of Schiff＇s Reagent 371

M117．Nucleal reaction of Feulgen and Rossenbeck 372

M118．Periodic acid-Schiff(PAS)reaction 373

M119．Peracetic acid-Schiff reaction 374

Concluding Comments on Histochemical Methods 375

Bibliography 377

Index 389