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

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
11. REFERENCES

Chapter 1 - GENERAL METHODS FOR ZEBRAFISH CARE

1. Availability and simple care
   
2. Food
   
3. Water
4. Fish Diseases
   • Velvet disease
   • Mycobacteriosis
   • Intestinal Capillariasis
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
   • Overview of in vitro fertilization
   • General Procedures and Overview
   • Squeezing Males
   • Squeezing Females
   • 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:
   • Embryo Extract
   • Growth Medium

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
   Heat shock
   Early pressure
6. Delayed In Vitro Fertilization Using Coho Salmon Ovarian Fluid
7. Production Of Androgenetic Haploids and Diploids
8. Freezing Sperm
9. Thawing And Using Frozen Sperm For In Vitro Fertilization
10. Fin Amputations
11. Gamma Ray Mutagenesis
12. Chemical Mutagenesis
13. Testosterone Treatment To Produce Males
14. 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