Open-book Preparations from Chick Embryos and DiI Labeling of Commissural Axons

Materials and Reagents

1. Fertilized eggs containing chicken embryos (incubated until Hamburger and Hamilton stage 25-26)
   
2. PBS (add 50 ml of 20x PBS to 950 ml of ddH₂O)
   
3. Sylgard silicone elastomer (World Precision Instruments, catalog number: SYLG184 )
   
4. Fast DiI (Life Technologies, Molecular Probes^®, catalog number: D-7756 ) (dissolved at 5 mg/ml in ethanol)
   
5. Vacuum grease (Dow Corning Corporation, catalog number: 976V )
   
6. 20x sterile phosphate buffered saline (PBS) (see Recipes)
   
7. 4% paraformaldehyde (4% PFA) (see Recipes)
   
8. Phosphate (PB) buffer (1 M, 1 L, pH 7.4) (see Recipes)
   

Equipment

1. Incubator set at 38.5 °C and 45% humidity (e.g. FIEM, Juppiter 576 Setter+Hatcher; Kendro Laboratory Products, Heraeus/Kendro, model: B12 )
   
2. Spring scissors (Fine Science Tools, catalog number: 15003-08 )
   
3. Dumont #5 forceps (Fine Science Tools, catalog number: 11252-20 )
   
4. 90 mm Petri dishes (e.g. Thermo Fisher Scientific, Sterilin^®, catalog number: 101VR20/C )
   
5. 0.20 mm insect pins (Fine Science Tools, catalog number: 26002-20 )
   
6. 0.10 mm insect pins (Fine Science Tools, catalog number: 26002-10 )
   
7. Microscope with adjustable transmitted light source (Leica MZ6 stereomicroscope with Leica CLS 150x )
   
8. Fine surgical scalpel (Grieshaber Logistic Group, catalog number: 68101 )
   
9. Tungsten wire (0.075 mm) (World Precision Instruments, catalog number: TGW0325 )
   
10. Metal flat spatula/spoon (e.g. Sigma-Aldrich, catalog number: S4022 , S3397 )
    
11. Borosilicate glass capillaries (outer Ø/inner Ø: 1.2 mm/0.68 mm) (World Precision Instruments, catalog number: 1B120F-4 )
    
12. Glass needle puller (Narishige Group, catalog number: PC-10 )
    
13. Polyethylene tubing (Ø 1.24 mm) (e.g. Angst and Pfister AG, catalog number: FT0110350018 )
    
14. Plastic transfer pipettes (e.g. Sarstedt, catalog number: 86.1171 )
    
15. 18 G x 1.5” needle (e.g. Braun, catalog number: 4665120 )
    
16. 1 ml syringe (e.g. Braun, catalog number: 9166017V )
    
17. 20 ml syringe (e.g. Braun, catalog number : 4606205V )
    
18. 24 mm x 24 mm glass coverslips (VWR International, catalog number: 631-0127 )
    
19. Microscope suitable for brightfield and fluorescent microscopy (e.g. Olympus, model: BX51 )
    

Procedure