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