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This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.

THE ZEBRAFISH BOOK
A guide for the laboratory use of zebrafish Danio (Brachydanio) rerio


by Monte Westerfield, Institute of Neuroscience, University of Oregon

  1. GENERAL METHODS FOR ZEBRAFISH CARE
  2. BREEDING
  3. EMBRYONIC AND LARVAL CULTURE
  4. MICROSCOPIC OBSERVATIONS
  5. CELLULAR METHODS
  6. DISSOCIATED CELL CULTURE
  7. GENETIC METHODS
  8. HISTOLOGICAL METHODS
  9. MOLECULAR METHODS
  10. RECIPES


Chapter 1 - GENERAL METHODS FOR ZEBRAFISH CARE

  1. Availability and simple care
  2. Food
  3. Water
  4. Fish Diseases
  5. Quarantine Room Procedures
  6. Cleaning Tanks
  7. Shipping Fish
  8. Keeping Track Of Stocks
  9. Zebrafish Standard Protocol Form

Chapter 2 - BREEDING

  1. Simple Method For Steady, Low-Level Embryo Production
  2. Method For Maximal Embryo Production
  3. Embryo Collection
  4. Simple Schedule For Breeding Zebrafish
  5. Zebrafish Breeding Schedule For Maximal Embryo Production
  6. Detailed Methods For Breeding Over Marbles
  7. Pair-Wise Breeding Of Individual Or Natural Cross Fish
  8. Embryo Production By In Vitro Fertilization

Chapter 3 - EMBRYONIC AND LARVAL CULTURE

  1. A Simple Method for Raising Babies
  2. Raising Larvae in a Nursery
  3. Simple, Quick Methods For Raising Paramecia
  4. A Semi-Sterile Method For Raising Paramecia
  5. Device for harvesting paramecia
  6. Growing Paramecia Under Sterile Conditions
  7. Paramecia Culture Medium
  8. Purification of Paramecia
  9. Microworms as a Food Source
  10. A Simple Method for Raising Larvae
  11. Developmental Staging Series

Chapter 4 - MICROSCOPIC OBSERVATIONS

  1. Removing Embryos From Their Chorions
  2. Viewing Chambers
  3. Agar Mounting
  4. Methyl Cellulose Mounting

Chapter 5 - CELLULAR METHODS

  1. A Device to Hold Zebrafish Embryos During Microinjection
  2. Blastomere Lineage Analysis
  3. DiI Labeling
  4. Labeling Single Cells With Lineage Tracers
  5. Laser-ablation of Single Cells
  6. Overview of Single Cell Transplantation
  7. Detailed Procedure for Transplanting Single Cells
  8. An Inexpensive and Easy Microinjection Embryo-Tray

Chapter 6 - DISSOCIATED CELL CULTURE

  1. Preparing Embryos for Cell Culture
  2. 0 Ca2+ - Mechanical Dissociation
  3. Protease Dissociation
  4. Dissociation of Embryos by Dissection
  5. Using Coated Glass Coverslips for Cell Culture
  6. Recipes:

Chapter 7 - GENETIC METHODS

  1. Conventions For Naming Zebrafish Genes
  2. Suggested Guidelines For Maintaining Mutant Stocks
  3. Overview Of Methods For Parthenogenesis
  4. Production Of Haploid Embryos
  5. Production Of Homozygous Diploid Embryos
  6. Heat shock
  7. Early pressure
  8. Delayed In Vitro Fertilization Using Coho Salmon Ovarian Fluid
  9. Production Of Androgenetic Haploids and Diploids
  10. Freezing Sperm
  11. Thawing And Using Frozen Sperm For In Vitro Fertilization
  12. Fin Amputations
  13. Gamma Ray Mutagenesis
  14. Chemical Mutagenesis
  15. Testosterone Treatment To Produce Males
  16. Genetic Strains

Chapter 8 - HISTOLOGICAL METHODS

  1. Preparing subbed slides for sections
  2. OCT Embedding for Cryostat Sectioning of Embryos or Larvae
  3. Agar Embedding for Cryostat Sectioning of Embryos or Larvae
  4. Gelatin Embedding for Vibratome Sectioning of Embryos or Larvae
  5. Staining Sections with Antibodies Using PAP
  6. Staining Whole-Mount Embryos with PAP
  7. Solutions for Antibody Staining Protocols
  8. Whole-Mount Staining of Biotin-Dextran Injected Zebrafish Embryos
  9. Photoconversion of Fluorescently Labeled Profiles for EM
  10. Chromosome Spreads
  11. Fixation by Freeze Substitution
  12. Zebrafish Monoclonal Antibodies

Chapter 9 - MOLECULAR METHODS

  1. Extraction Of Proteins From Zebrafish Embryos For SDS-Gel Analysis
  2. Western Blots Of Zebrafish Embryos
  3. Preparation Of Genomic Dna
  4. Preparation Of Genomic Dna For Southern Analysis
  5. Rapid Isolation Of Rna From Zebrafish
  6. Extraction And Purification Of Rna From Zebrafish Embryos
  7. Preparation Of High Quality Rna From Zebrafish Embryos
  8. Whole-Mount In Situ Hybridization
  9. High Resolution Whole-Mount In Situ Hybridization
  10. Two Color Whole-Mount Rna In Situ Hybridization
  11. Sections Of Whole Mount In Situ Hybridization Preparations
  12. Total Nucleic Acid Extraction Procedure For Zebrafish Embryos
  13. A Simplified Ribonuclease Protection Assay For Zebrafish Embryos

Chapter 10 - RECIPES

  1. ABC Solution (Avidin-Biotin HRP Complex)
  2. Agar/Sucrose
  3. Atabrine Stock Solution
  4. Blocking Solution for Western Blots
  5. BSA/PBS/DMSO
  6. BT Fix
  7. Chorion Removal
  8. DAB (Diaminobenzidine)
  9. DAB Heavy Metal Stain
  10. DAB Presoak Solution
  11. DAB Solution
  12. DNA Extraction Buffer
  13. EDTA
  14. Egg Water
  15. Embryo Extract
  16. Embryo Medium
  17. Epon
  18. Epon/Araldite
  19. Finquel (MS222)
  20. Fish Water
  21. Fix Buffer
  22. 1.25X Fix Buffer
  23. Fixatives
  24. Gelatin Embedding Medium
  25. Genomic DNA Extraction Buffer
  26. GHCl Buffer
  27. Giemsa
  28. Ginzburg Fish Ringers
  29. GIT Buffer
  30. Growth Medium
  31. Hank's (Final)
  32. Hank's (Full Strength)
  33. Hank's Premix
  34. Hank's Stock Solutions
  35. Heat Shock (Diploid eggs)
  36. In situ Hybridization Staining Buffer
  37. Paramecia Seed Cultures
  38. Paramecia for Baby Fish (Traditional Method)
  39. Paramecia for Baby Fish (Streamlined Procedure)
  40. Paramecia Medium Stock Solution I
  41. Paramecia Medium Stock Solution II
  42. Paramecia Storage Medium (Long Term)
  43. PBS
  44. 2X PBS
  45. PBS/BSA/DMSO
  46. PCR Extraction Buffer
  47. PMSF
  48. PO4 buffer (0.1 M, pH 7.3)
  49. Pronase
  50. Protein Extraction Buffer
  51. PTU
  52. Ringer's Solutions
  53. Salt Stock
  54. SDS Sample Buffer
  55. Schönefeld's Medium for Growing Paramecia
  56. Sodium Bicarbonate
  57. Sperm Freezing Medium
  58. Sperm Thawing Medium
  59. Stock salts
  60. Subbing Solution for Slides
  61. TBS
  62. TES buffer
  63. Tricaine
  64. TTBS
  65. Western Blot color development buffer
  66. Western Blot color development solution
  67. Whole-Mount in situ Hybridization Solutions