Materials and Reagents

1. Petri dish, 100 mm diameter (Greiner Bio-One, catalog number: 627102)

2. 12/24 well plate (Corning, catalog numbers: 3512 [12-well]; 3527 [24-well])

3. Agarose coated dishes (homemade): 10 cm diameter, 6 cm diameter (Greiner Bio-One, catalog numbers: 627102 [10 cm diameter]; 628102 [6 cm diameter]) and 6-well plates (Roth, catalog number: ATCO.01). For the coating, use agarose 1% in ERM (medaka) or E3 (zebrafish). The base of the plastic plate should be covered by agarose; 1-2 mm thick is enough.

4. Cell saver tips (200 μl) (Biozyme, catalog number: 729051)

5. Borosilicate glass capillaries, 1.2 mm outer diameter × 0.94 mm internal diameter (Mind that these are different from the injection needles. They should NOT contain an internal filament. HARVARD Apparatus, model: 30-0016 GC120T-10 or GC120T-15)

6. Razor blade (to cut open the borosilicate needle) (Schreiber Instrumente, catalog number: 11-0240)

7. For Homemade (Air) transplantation device

   1. Tube connector, Syringe Mouthpiece (Narishige, model: CI-1)

   2. Teflon Tubing (Narishige, model: CT-1)

   3. Handle Probe (WPI, catalog number: 2505)

   4. Needle Holder (2 MM Press Port 1.2 MM, WPI catalog number: MPH412)

   5. Epoxy Potting (Gray, WPI, catalog number: 4886)

   6. 1 ml Syringe (BD Plastipak, catalog number: REF300013)

8. Glass Pasteur pipette (WU Mainz, catalog number: 200760)

9. Glass Pasteur pipette, opened tip

10. Glass plates (40 mm diameter; Roth, catalog number: T937.1)

11. Forceps (Dumont No. 5 or No. 55)

12. Long tips – to handle medaka embryos after dechorionation (Eppendorf, model: 5242956003, Microloader 20 µl in Racks)

13. Sandpaper (240/180)

14. Plastic Pasteur pipette (Biosigma, catalog number: 390512)

15. Sieve to wash zebrafish embryos

16. Net to collect medaka females (homemade)

17. Hook to collect medaka embryos (homemade, see details in Figure 1)

    
    

    
    Figure 1. Hook to collect medaka embryos. Inoculation loop holder (Roth, KL97.1), length 21 cm; wire 1.4301: length 2 cm, width 2.5 cm.

    
    

18. Glass flasks (100 ml, 500 ml)

19. 100 ml Beakers (Schott, catalog number: 211062402)

20. Low melting point agarose (Roth, catalog number: 6351.5)

21. Pronase (Roche, catalog number: 10165921001)

22. Hatching enzyme (HE) medaka (home-made, can be ordered at NBRP Medaka: https://shigen.nig.ac.jp/medaka/strain/hatchingEnzyme.jsp;jsessionid=8F08DDE7593CDD7D1BFA32E6382E7E0F)

23. Transplantation mold (we use homemade molds; see Figure 2. Similar commercial molds can be found at: https://www.wpi-europe.com/products/pumps-and-microinjection/oocyte-injection/z-molds.aspx)

24. Embryo rearing medium for medaka (ERM) (see Recipes)

25. Zebrafish medium (see Recipes)

26. 20× Tricaine (Sigma-Aldrich, catalog number: A5040-100G) (See Recipes)

    
    
    Figure 2. Transplantation molds. To create the transplantation plate, molds are inserted top-down in 1.5% Agarose (see section B1). (A) Petri dish filled with 1.5% agarose in water, ca. 60°C. (B-B’) Transplantation molds with a detailed view of the structures that will form the wells. (C) Transplantation molds are placed in the Petri dish containing agarose, with the wells facing the agarose. (D) Transplantation mold stays in the Petri dish until the agarose has formed a stable gel. (E) When the transplantation mold is removed, the agarose gel contains wells in which the embryos will be placed. The transplantation plate is ready then.

Equipment

1. Zebrafish housing system (Tecniplast ZEBTEC equipped with 3.5 L tanks)

2. Zebrafish mating boxes (homemade 5 L boxes; see Figure 3. Alternatively, we have used breeding tanks from Tecniplast – 0.8 or 1.7 L, sloped tank)

   
   

   
   Figure 3. Zebrafish mating box. Lateral view (top) and top view (bottom) of the zebrafish mating box. This setup accommodates up to five couples.




3. Medaka housing system (Tecniplast ZEBTEC equipped with 3.5 L tanks and Müller & Pflege, 4 L tanks)

4. Incubator, 23°C (no need of CO₂ control) (Binder, model: 9020-0339)

5. Incubator, 28°C (no need of CO₂ control) (Rumed, model: 200330160)

6. Incubator, 32°C (no need of CO₂ control) (Binder, model: 9010-0081)

7. Micropipette puller (we use a horizontal puller, Sutter Instrument CO, model: P-97)

   To pull Harvard Apparatus GC120T – 10 capillaries, we use the following program: Heat 505; Pull 25; Vel 250; Time 10)

8. Transplantation device (a homemade air system; see Figure 4. We have also successfully used the Eppendorf CellTram^R Air/Oil system)

   
   

   
   Figure 4. Set up for Transplantations. Main components: (1) Micromanipulator and Stereoscope with light source; (2) transplantation needle inserted into needle holder; (3) tube attached to the needle and a syringe (airtight); (4) syringe (volume 1 ml) attached to desk.




9. Stereoscope with light source (Olympus, model: SZX7)

10. Micromanipulator (Eppendorf, model: InjectMan NI 2)

11. Pipette (Gilson 20-200 µl, 200-1,000 µl)

12. Fluorescence stereomicroscope (Nikon, model: SMZ18, solar light engine from Lumencor)

13. Microwave

Procedure