Materials and Reagents

1. hTERT-RPE1 cell line (ATCC , catalog number: CRL-4000 )
   
2. Zebrafish (AB strain) (China Zebrafish Resource Center)
   
3. DMEM/F12 medium (Life Technologies, Invitrogen^TM, catalog number: 11330-032 )
   
4. Fetal bovine serum (Life Technologies, Invitrogen^TM, catalog number: 16000-044 )
   
5. Hygromycin B (Life Technologies, Invitrogen^TM, catalog number: 10687010 )
   
6. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
   
7. Pure ethanol (Sinopharm Chemical Regent, catalog number: 10009218 )
   
8. Low-melting-point agarose (Sigma-Aldrich, catalog number: A9414 )
   
9. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
   
10. Triton X-100 (AMRESCO, catalog number: 0694 )
    
11. Tween-20 (AMRESCO, catalog number: 0777 )
    
12. Mouse monoclonal anti-acetylated tubulin antibody (Sigma-Aldrich, catalog number: T-6793 )
    
13. Goat anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-546 (Life Technologies, catalog number: A11030 )
    
14. Donkey anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-488 (Life Technologies, catalog number: A21202 )
    
15. 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D8417 )
    
16. Dako mounting medium (Dako, catalog number: s3023 )
    
17. PBS (see Recipes)
    
18. PBT (see Recipes)
    
19. 4% paraformaldehyde (PFA) fixation solution (see Recipes)
    
20. Embryo fixation buffer (see Recipes)
    
21. Embryo blocking buffer (see Recipes)
    
22. 4', 6-diamidino-2-phenylindole (DAPI) stock solution (see Recipes)
    
23. Complete growth medium (see Recipes)
    
24. Serum free medium (see Recipes)
    
25. 75% ethanol (see Recipes)
    

Equipment

1. Millex-GP Filter unit with 0.22 µm pore size (Millipore, catalog number: SLGP033RS )
   
2. Forceps (The duMONT Company, catalog number: 0203-5/15-PO )
   
3. 10 cm Petri dishes (BD Biosciences, Falcon^®, catalog number: 353003 )
   
4. 12-well plates (Corning, catalog number: 3513 )
   
5. 2 ml Eppendorf (EP) tubes (Axygen, catalog number: MCT-200-C )
   
6. Glass slides (Fan Yi, catalog number: 7105P )
   
7. 18mm diameter circle cover slips (Thermo Fisher Scientific, catalog number: 12-545-84 )
   
8. Parafilm (Parafilm M^®, catalog number: PM-996 )
   
9. 100 ml beaker (Thermo Fisher Scientific, catalog number: 1201-0100 )
   
10. Filter paper (GE, catalog number: 10311611 )
    
11. Plastic transfer pipette (Thermo Fisher Scientific, catalog number: 11387873 )
    
12. Shakers (Qilinbeier, catalog number: TS-8S )
    
13. Leica TCS SP5 confocal microscope equipped with a 63x oil objective and a 63x NA1.2 water immersion objective
    

Procedure