Cerebellum Dissection and Slice Preparation: A Method to Generate Tissue Slices from Mouse Cerebellum

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

1. Cerebellum Dissection and Sections Preparation (Hands-on Time ≈ 15–20 min per animal)

1. Place one of the 60 mm cell culture dishes containing the ice-cold dissection medium under the binocular microscope and remove the top plate.
2. Swab the head fur with 70% ethanol (carefully avoiding touching the eyes of the animal) and decapitate the animal using large sharp scissors, according to the approved procedure.
   NOTE: The cerebellar slices are generated from P9-P10 mice without any specific sex or strain bias.
3. Use small scissors to cut the skin along the midline of the head starting from the neck and going up to the mouse muzzle.
4. To hold the head and expose the skull, fold back the skin ventrally under the head, and pinch the two folds of skin between one’s fingers.
5. Insert the small scissors gently into the foramen magnum and cut the skull by making one lateral incision towards the side and then cut all around the head skull. Keep the tip of the scissors as parallel and close to the skull as possible while cutting, to avoid damaging the brain tissue.
6. Retrieve the dorsal part of the skull using fine-straight forceps. While carefully lifting the dorsal part of the skull, cut the adhering meninges (translucent irrigated membrane surrounding the brain tissue) if needed with small scissors to avoid damaging the brain tissue.
7. Carefully introduce fine-straight forceps (or alternatively use a spatula) between the ventral skull and the brain, gently flip out the brain and cut the optic and trigeminal nerves with small scissors.
8. Help the brain to drop by gravity into the 60 mm cell culture dish containing ice-cold dissection medium by turning the head upside down just above the dish and release the last adhesions with small scissors if needed.
9. Using fine forceps, orientate the brain with the dorsal side facing the researcher, and the ventral side lying on the bottom of the dish.
10. Under the binocular microscope, use the fine-straight forceps to immobilize the brain on the forebrain side and avoid touching the hindbrain, as it could get damaged. Separate the hindbrain from the rest of the brain (fore- and midbrain, see schematic on Figure 1Aa), using fine-straight forceps. To separate the cerebellum from the rest of the hindbrain, use the fine forceps to cut the cerebellar peduncles underneath the cerebellum (see schematic on Figure 1Aa’).
11. Once the cerebellum is isolated, carefully tear away the meninges using fine-straight forceps (see schematic on Figure 1Aa’).
12. Hold the cerebellum gently with the fine curved forceps and place it with the dorsal side up onto the plastic platform perpendicularly to the chopper razor blade (see schematic on Figure 1Bb).
13. Aspirate any excess of dissection medium around the cerebellum using a sterile thin-end pipette tip adapted on a 1 mL pipette (see schematic on Figure 1Bb).
14. Ensure that the cerebellum lays flat, but is not stretched, once placed onto the platform to get optimal sagittal slices during sectioning (see schematic on Figure 1Bb).
15. Slice 300 μm-thick sagittal sections of the cerebellum with the tissue chopper (see schematic on Figure 1Bb’). The force and speed of the blade have to be optimized on site.
16. Gently add a drop of dissection medium onto the sliced cerebellum. Using a wide-bore pipette tip placed onto a 1 mL pipette, slowly aspirate the sliced cerebellum and transfer it back into the 60 mm cell culture dish containing ice-cold dissection medium (see schematic on Figure 1Bb’’).
17. Clean the tissue chopper’s plastic platform with 100% ethanol between each cerebellum slicing. Let the ethanol evaporate before placing the next cerebellum onto the platform.
18. Use two fine-straight forceps (or alternatively titanium needles) to separate individual slices and select 6–8 slices from the vermis (see schematic on Figure 1Cc and 1Cc’).
19. Take a 6-well plate with culture inserts and transfer up to 4 selected slices onto one culture insert along with some dissection medium, using a wide-bore pipette tip adapted on a 1 mL pipette (see schematic on Figure 1Cc’).
20. Place the slices in the middle of the culture insert using the dissection medium or forceps to push and pull them gently into the right location, avoiding them to be in contact with each other (see schematic on Figure 1Cc’).
21. Remove any excess of dissection medium around the slices using a thin-end pipette tip. Ensure that the slices lay flat on the membrane (see schematic on Figure 1Cc’).
22. Place the 6-well plate into the incubator immediately after having added the slices on the membrane.

Figure 1: Illustration of cerebellum slices generation. The dissection is divided in three main steps. (A) The cerebellum is isolated, and the meninges removed (steps 1.10 and 1.11). (B) The cerebellum is then transferred to the chopper platform and sliced (steps 1.12 to 1.16). (C) Lastly, the slices are isolated from the vermis and placed on a membrane insert. The dissection medium transferred with the slices is removed and the 6-well plate is placed into the incubator (steps 1.18 and 1.22).