Alcohol: Methods and Protocols

Cover......Page 1
Frontmatter......Page 2
1 Introduction......Page 20
2.2 Oral Gavage......Page 21
3.1 Intraperitoneal Injection......Page 22
3.2 Oral Gavage......Page 24
4 Notes......Page 25
References......Page 26
A Voluntary Oral-Feeding Rat Model for Pathological Alcoholic Liver Injury......Page 27
2.2 Assessment of Urine Alcohol......Page 28
2.6 Immunohistochemical Analysis......Page 29
2.9 Electrophoretic Mobility Shift Assay......Page 30
2.11 Western Blotting......Page 31
2.13 Chymotrypsin (2)......Page 33
3.1.2.1 Processing of Blood, Liver Samples, and Histological Sections......Page 34
3.1.2.4 Hematoxylin and Eosin (H&E) Staining......Page 35
3.2 Biochemical assay of ALT......Page 36
3.3.1 Assay on Serum total 8-Isoprostane Level......Page 37
3.3.2 Thiobarbituric Acid Reactive Substances (TBARS)......Page 38
3.3.6 Immunohistochemical Analysis......Page 39
3.3.7.1.1 Total RNA Extraction......Page 40
3.3.7.1.3 Preparation of Complementary DNA From RNA......Page 41
3.3.7.2.1.3 Protein Assay......Page 42
3.3.7.2.1.4 Measurement of Protein Expression With Western Blotting......Page 43
3.3.8 Analysis of the DNA-Binding Activity of the Transcription
Factors (NF-κB and AP-1) With EMSA......Page 44
References......Page 46
Intragastric Ethanol Infusion Model in Rodents......Page 48
1 Introduction......Page 49
2.2 Diet......Page 50
3.1.1 Rat Intragastric Catheter......Page 54
3.1.2 Mouse Intragastric Catheter......Page 55
3.2 Infusion Set-up......Page 56
3.3 Diet Preparation......Page 57
3.4.2 Mouse Surgery......Page 58
3.5.1 Rat Intragastric Infusion......Page 59
3.6 Animal Monitoring......Page 60
References......Page 62
A Practical Method of Chronic Ethanol Administration in Mice......Page 64
1 Introduction......Page 65
3.1 Overview of Ethanol Administration Methods in Mice......Page 66
3.2.2 Troubleshooting......Page 67
3.4.1 Labeling......Page 68
3.4.2 Ethanol Preparation and Administration......Page 69
3.5.1 Weight Gain and Practical Starting Weights......Page 70
3.7.1 Weight Gains......Page 72
4 Notes......Page 73
References......Page 74
Analysis of Ethanol Developmental Toxicity in Zebrafish......Page 76
1 Introduction......Page 77
3 Methods......Page 78
3.3 Embryonic Ethanol Dose Determination......Page 79
3.4.1 In Vivo Cell Death......Page 80
3.4.2 Apoptotic Cell Death With TUNEL Analysis......Page 81
3.5 Global Gene Expression Analysis......Page 82
3.6 Temporal and Spatial Gene Expression Analysis......Page 83
3.7 Transient Gene Repression Techniques......Page 84
References......Page 85
The Avian Embryo in Fetal Alcohol Research......Page 88
2.2 Ethanol Treatment......Page 89
2.5 Ratiometric Imaging of Cai 2+ With Fura-2......Page 90
3.1 Egg Handling......Page 91
3.3 Pharmacologic Manipulation of Embryos With Microbead Implants......Page 92
3.4 Apoptosis Assessment......Page 93
3.5 Ratiometric Imaging of Cai 2+ With Fura-2......Page 94
4 Notes......Page 95
References......Page 97
Artificial Rearing......Page 98
1 Introduction......Page 99
2.1.2 Treatment Diet......Page 100
2.4 Artificial Rearing Components......Page 101
3.1.1 Stock Milk Diet......Page 103
3.2.1.2 Intragastric Cannula......Page 104
3.2.1.3 Washers......Page 105
3.2.2 Gastrostomy Surgery......Page 106
3.3 Artificial Rearing......Page 108
3.3.2 Determination of Diet Infusion Rate......Page 109
3.3.4 Care of Artificially Reared Pups......Page 110
4 Notes......Page 111
References......Page 113
Intragastric Intubation of Alcohol During the Perinatal Period......Page 114
1 Introduction......Page 115
2.3 Measurement of Blood Alcohol Concentrations (BACs)......Page 117
3.1 Alcohol Administration During the Prenatal Period......Page 118
3.3 BACs......Page 120
4 Notes......Page 121
References......Page 122
Human Monocytes, Macrophages, and Dendritic Cells......Page 125
1 Introduction......Page 126
2.3 Electrophoretic Mobility Shift Assay for Assaying NF-κB......Page 127
3.1 Isolation of Peripheral Blood Monocytes......Page 128
3.2 Generation of Human Monocyte-Derived Macrophages......Page 129
3.4 Isolation of Plasmacytoid DCs (PDCs)......Page 130
3.7 Cell Stimulation With Pathogen-Derived Agents......Page 132
3.9 Isolation of Nuclear Proteins......Page 133
3.11 Mixed Lymphocyte Reaction......Page 134
References......Page 135
Generation and Use of Primary Rat Cultures for Studies of the Effects of Ethanol......Page 137
2.2 Organotypic Slice Cultures......Page 138
3.2.1 Surgical Equipment......Page 139
3.2.3.2 Dissection of the Rat Cortices......Page 140
3.3.1 Reagents......Page 141
3.4.1 Equipment......Page 142
3.4.3.2 Preparation and Culture of Mouse Cortical Slices......Page 143
3.5.1 Equipment......Page 144
4.1 Fresh Samples......Page 145
5 Notes......Page 146
References......Page 147
Development and Properties of HepG2 Cells That Constitutively Express CYP2E1......Page 149
1 Introduction......Page 150
2.3 Cell Toxicity......Page 151
2.7 Caspase Activity......Page 152
3.1 Cell Culture and Storage......Page 153
3.3 MTT Assay to Determine Cytotoxicity......Page 154
3.6 Lipid Peroxidation Analysis......Page 155
3.8 Measurement of GSH Levels......Page 156
3.11 Apoptosis Analysis by Determination of DNA Fragmentation (DNA Ladder)......Page 157
3.14 Activation of p38 MAPK......Page 158
4 Notes......Page 159
References......Page 161
Modeling the Impact of Alcohol on Cortical Development in a Dish......Page 163
1.1 The Fetal Alcohol Syndrome......Page 164
1.2 Effects of Alcohol During Brain Growth and Development......Page 165
1.3 Neurodevelopment During the Second-Trimester Period......Page 166
1.4 The Neurosphere Culture Model Recapitulates the Second-Trimester Neuroepithelium......Page 167
1.5 Mapping the Diversity of Fetal Neuroepithelial Cells......Page 168
1.6 Modeling Early Cortical Neuronal Differentiation With Culture Models......Page 169
2 Materials......Page 170
3.2 Ethanol Treatment......Page 171
3.3 Differentiation of Neurosphere Cultures......Page 172
4 Notes......Page 174
References......Page 176
Acetaldehyde-Induced Barrier Disruption and Paracellular Permeability in Caco-2 Cell Monolayer......Page 181
1 Introduction......Page 182
2.4 Measuring Paracellular Permeability......Page 183
2.5 Immunofluorescence Staining and Confocal Microscopy......Page 184
3 Methods......Page 185
3.3 Measuring TER......Page 186
3.4 Measuring Paracellular Permeability......Page 187
3.5 Immunofluorescence Staining and Confocal Microscopy......Page 189
3.6 LDH Release Assay......Page 190
4 Notes......Page 191
References......Page 192
Alcohol-Induced Oxidative Stress in the Liver In Vivo Measurements......Page 194
1 Introduction......Page 195
2.1 Generation of an In Vivo Positive Control for Staining......Page 196
2.3 Immunohistochemistry......Page 197
3.2 Harvesting Tissue for Immunohistochemistry......Page 198
3.3 Deparaffinization and Rehydration of Tissue Slides......Page 200
3.5 Immunohistochemical Detection of Haptene Signals......Page 201
3.7 Image Analysis of Immunohistochemistry......Page 202
4 Notes......Page 204
References......Page 205
Isolation of Kupffer Cells From Rats Fed Chronic Ethanol......Page 207
2.3 Isolation and Culture of Kupffer Cells......Page 208
3.1 Setting up the Peristaltic Pump and Perfusion......Page 210
3.3 Setting Up the Tissue Culture Room......Page 212
3.5 Surgery......Page 213
3.6 Isolation of Kupffer Cells......Page 214
4 Notes......Page 218
References......Page 220
Dendritic Cells in Chronic In Vivo Ethanol Exposure Models......Page 221
1.3 Effect of Chronic EtOH Exposure on DCs......Page 222
2.1.1 Purification of CD8a+ and CD8a- DCs From the Spleen......Page 223
2.1.3 Purification of Pulmonary DC Subsets by FACS......Page 224
2.2.1 Immunofluorescent Staining of LCs in Epidermal Sheets......Page 225
2.2.3 Assessment of rDC Migration From the Lungs to the Lymph Nodes by i.n. CFSE Labeling......Page 226
2.3.1.2 CD8 T Cell Activation......Page 227
2.3.2 DC Activation of Antigen-Specific T-Cell Proliferation In Vivo......Page 228
3.1.1 Purification of Mature Bulk Splenic DCs......Page 229
3.1.3 Manual MACS Purification of Mature CD8a- and CD8a+ DCs From Splenocytes......Page 230
3.1.5 Purification of Pulmonary DC Subsets by FACS......Page 231
3.2.1 Preparation and Fixation of Epidermal Sheets......Page 232
3.2.1.1 Staining the Epidermis for LCs......Page 233
3.2.2.2 Intracellular Staining......Page 234
3.2.3 Assessment of rDC Migration From the Lungs to the Lymph Nodes by i.n. CFSE Labeling......Page 236
3.3.1 DC Activation of Antigen-Specific T-Cell Proliferation In Vivo......Page 237
3.3.2 Allostimulatory Assay of DC Function......Page 238
4 Notes......Page 239
References......Page 240
Formation and Immunological Properties of Aldehyde-Derived Protein Adducts Following Alcohol Consumption......Page 242
1 Introduction......Page 243
2.1 Preparation of 5X Chelator Solution......Page 244
2.3 Synthesis of Hexyl-MAA......Page 245
2.5 Preparation of Lysine Sepharose 4B-CL Beads......Page 246
2.6 Method of Habeeb for Determining Lysine Modification (30)......Page 247
2.11 Peroxidase Substrate for ELISAs......Page 248
2.13 Antibodies to Aldehydes (see Note 1)......Page 249
2.15 Western Blot for the Detection of Adducts......Page 250
3.1 Generation of Malondialdehyde-Acetaldehyde Adducts......Page 251
3.3 Generation of Reduced Acetaldehyde Adducts......Page 252
3.4 Generation of Nonreduced Acetaldehyde Adducts......Page 253
3.6 Determination of Macromolecule Modification by 14C Acetaldehyde......Page 254
3.8 Absorption and Elution of Polyclonal Antibodies From Sepharose 4B-CL Lysine Beads Modified with Nonreduced AA Adducts, Reduced AA Adducts, or MAA Adducts......Page 255
3.9 Measurement of Circulating Antibody Responses in Mice by ELISA......Page 256
3.12 Analysis of the Antibody Specificity by Competitive ELISA......Page 257
3.13 T-Cell Proliferation Assays......Page 258
3.15 Immunoprecipitation of Adducted Macromolecules......Page 259
3.17 Western Blot for the Detection of Adducts......Page 260
References......Page 262
Assessment of Natural Killer (NK) and NKT Cells in Murine Spleens and Livers......Page 265
1 Introduction......Page 266
2.2 Flow Cytometry Examination of Surface and Cytoplasmic Markers......Page 267
3.1.2 Isolation of Hepatic Mononuclear Cells......Page 268
3.2.1 Flow Cytometry Staining for Cell-Surface Molecules......Page 269
3.2.2 Intracellular Staining for IFN-γ......Page 270
3.3 Functional Assessment of NK and NKT Cells......Page 272
3.3.3 Plating (Typically Performed in Duplicates)......Page 273
3.3.4 Data Analysis......Page 274
3.4 Cytokine Production......Page 276
3.5 Stimulation of NKT Cells by α-GalCer......Page 277
4 Notes......Page 279
References......Page 280
Polyclonal and Antigen-specific Responses of T Cells and T Cell Subsets......Page 283
1 Introduction......Page 284
2.3 Flow Cytometry Staining......Page 285
2.5 Antigen-Specific T Cells, Using Attenuated, L. Monocytogenes as a Model Organism......Page 286
3.1 Isolation of T Cells From Spleen, Thymus, or Lymph Node......Page 287
3.2.1 Column Purification of CD4+ T Cells by Negative Selection......Page 288
3.2.4 B-Cell Depletion of Splenocytes......Page 289
3.4 Stimulation and Cytokine Response of Cells After Polyclonal Stimulation......Page 290
3.5 Antigen-Specific T Cells, Using Attenuated, L. Monocytogenes as a Model Organism......Page 291
3.5.2 Cell Preparation......Page 292
3.5.4 Antigen Presenting Cells (APCs)......Page 293
3.5.5 Intracellular Staining for Cytokines after Brefeldin A Treatment......Page 294
3.6 Time Course of Response......Page 295
3.7.1 Adoptive Transfer Utilizing Congenic Strains of Mice......Page 296
3.7.2 Fluorescent-labeled MHC Class I Pentamers and MHC Class II Tetramers......Page 297
4 Notes......Page 298
References......Page 299
B-Cell Studies in Chronic Ethanol Mice......Page 301
1 Introduction......Page 302
2.2.1 MACS Bead Enrichment......Page 303
2.2.5 B-Cell Activating Agents......Page 304
2.3.2 Adjuvants......Page 305
2.3.4 TNP-Specific ELISA......Page 306
3.1 Physical Assessment of the B-Cell Compartment in Mice......Page 307
3.1.1 Flow Cytometric Analysis of B Cells From Bone Marrow, Spleen, Lymph Nodes, and Peritoneal Cavity......Page 308
3.1.2.1 Tissue Freezing......Page 310
3.1.2.3 Tissue Staining......Page 311
3.2.1 MACS Bead Enrichment of B Cells......Page 312
3.2.2 Culturing of B Cells......Page 313
3.2.4. Apoptosis of B Cells......Page 314
3.2.5 Differentiation of B Cells......Page 315
3.4 Immunization Procedures......Page 316
3.4.1.1 Preparation of Alum [Al(OH)3] Slurry......Page 317
3.4.2.1 ELISA for TNP-Based Antigens......Page 318
3.4.2.1.1 Preparation of TNP-HGG-Biotin......Page 319
3.4.2.2 ELISA for TNP-Specific IgM, IgG1, IgG2a, IgG3, and IgE......Page 320
3.4.2.3 ELISA for NP-Specific IgG1......Page 321
3.4.2.3.2 NP ELISA......Page 322
3.4.3. Flow Cytometric Assays for GC and Memory B Cells......Page 323
3.4.3.1.2 Preparation of PE for Immunization......Page 324
3.4.3.2.1 Detection of PE-Specific Murine Memory B Cells With Flow Cytometry......Page 325
4 Notes......Page 326
References......Page 327
Histological Analysis of Bone......Page 330
1 Introduction......Page 331
2.3 Fixation......Page 332
2.6 Staining......Page 333
3.1 Fluorochrome Labeling......Page 334
3.3 Fixation......Page 336
3.4 Embedding......Page 337
3.5 Sectioning Undecalcified Bone......Page 338
3.6 Staining......Page 339
3.7 Ground Cortical Sections......Page 340
3.8 Histomorphometry......Page 341
3.9 Conclusion......Page 343
4 Notes......Page 345
References......Page 346
Assessing Effects of Alcohol Consumption on Protein Synthesis in Striated Muscles......Page 347
2 Materials......Page 348
3 Methods......Page 349
3.1 Measurement of Protein Synthesis In Vivo in Rats......Page 350
3.4 Dabsylation of Plasma Amino Acids......Page 352
3.6 Measurement of Incorporation of Radioactivity into Muscle Protein......Page 353
3.7.2 Enrichment of Myofibrillar Protein Fraction......Page 354
3.8 Incubated Epitrochlearis Preparation......Page 355
3.9 Discussion......Page 356
4 Notes......Page 357
References......Page 358
Methods to Investigate the Effects of Chronic Ethanol on Adipocytes......Page 360
2.1 Adipocyte Isolation......Page 361
2.3 Adiponectin Secretion......Page 362
3.1 Adipocyte Isolation......Page 363
3.2.1 Assay......Page 364
3.3 Adiponectin Secretion......Page 365
3.4 Adipocyte Lipolysis......Page 366
4 Notes......Page 367
References......Page 368
Proteomic Approaches to Identify and Characterize Alterations to the Mitochondrial Proteome in Alcoholic Liver Disease......Page 371
2.1 Isoelectric Focusing......Page 372
2.2.2 Two-Dimensional Blue Native-Polyacrylamide Gel Electrophoresis......Page 373
3 Methods......Page 374
3.2 Running One-Dimensional Isoelectric Focusing Gels......Page 376
3.3 Running 2-D IEF/SDS-PAGE Gels......Page 377
3.4 Running 1-D BN-PAGE Gels......Page 378
3.5 Running 2-D BN-PAGE Gels......Page 379
3.6 Image Analysis of Gels......Page 380
4 Notes......Page 381
References......Page 382
Alcoholic Liver Disease and the Mitochondrial Ribosome......Page 383
1 Introduction......Page 384
2.1 Isolation of Mitochondrial Ribosomes From Mitochondria......Page 385
2.4.2 Acetic Acid-Acetone Procedure......Page 386
2.6 Equipment......Page 387
3.1 Isolation of Mitochondrial Ribosomes From Mitochondria......Page 388
3.3 Preparation of Soluble Mitochondrial Translation Factors......Page 390
3.4.2 Acetic Acid-Acetone Procedure......Page 391
3.5 2D-Electrophoretic Analyses Of Mitochondrial Ribosomal Proteins (NEPHGE)......Page 392
4 Notes......Page 395
References......Page 396
Microarray Analysis of Ethanol-Induced Changes in Gene Expression......Page 397
1 Introduction......Page 398
2.1 DNA Microarrays......Page 399
3.1 Experimental Design......Page 400
3.2 Protocol......Page 403
3.3.1 Affymetrix-Specific Quality Control Metrics......Page 404
3.4 Primary Analysis......Page 405
3.5 Statistical Filtering......Page 406
3.6 Multivariant Analysis......Page 407
3.7 Bioinformatics and Network Analysis......Page 408
4 Notes......Page 409
References......Page 410
Index......Page 413