Obtaining Cerebellum Slices from an Embryonic Chick Brain

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissection of Chicken Embryo Day 14 (e14) Cerebellum

1. Incubate brown fertilized hens' eggs at 38 °C to embryonic Day 14.
2. Using egg scissors, decapitate the chick embryo in ovo and remove the head to a Petri dish containing ice-cold Phosphate buffer saline (PBS) (Figure 1A).
3. Using standard forceps, make incisions behind each eye all the way through the tissue, removing the eyes and upper jaw. Make a second incision all the way through the pharynx, removing the lower jaw (Figure 1B).
4. Using standard forceps, remove all skin from the surface of a skull by peeling it away (Figure 1C) and remove frontal and parietal bones, revealing the brain.
5. From a ventral aspect, remove pharyngeal cartilage and auxiliary mesenchyme.
6. From a dorsal aspect, carefully remove the mesenchyme dorsal to the hindbrain, taking care not to damage pia, and separate the entire brain (Figure 1D).
7. Make an incision all the way through the tissue between the midbrain and hindbrain and separate the hindbrain, including the cerebellum (Figure 1E).
8. Remove the entire cerebellum by making incisions all the way through the tissue at both lateral junctions of the cerebellum and alar plate of the hindbrain, taking care to maintain the integrity of the pia throughout dissection (Figure 1F). Also, remove the forming choroid plexus (Figure 1G).
9. Move the dissected cerebellum into ice-cold Hank's balanced salt solution (HBSS).

2. Slice Culture of e14 Cerebellum

1. Transfer the whole cerebellum to the sterile platform of a Tissue Chopper using a spatula or a 3 ml Pasteur pipette with the tip cut away to widen the aperture. Remove excess liquid using a pipette.
2. Cut entire whole cerebellum in the required orientation at 300 µm thickness using the tissue chopper set with a cutting speed at 50% of the maximum. Tissue integrity is most easily preserved in sagittal section; however, orientation is also an important consideration for cell analysis (Purkinje cell dendrites are sagittally aligned, while granule cell axons run transversely, perpendicular to the plane of Purkinje cell dendrites).
3. Using a 3 ml Pasteur pipette, cover the sliced cerebellum in ice-cold HBSS.
4. Transfer the slices in HBSS to a 60 mm Petri dish containing ice-cold fresh HBSS using a 3 ml Pasteur pipette with a cut tip.
5. Under a dissecting microscope illuminated with a fiber optic light source, ensure separation of individual slices using watchmaker forceps.
   Note: Each dissection, from embryo to slice incubation in culture medium, takes around 10-20 min, depending upon experience. Perform dissections one at a time to ensure that the cerebella spends the minimum amount of time between decapitation and ex vivo culture.