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

In mice, the cerebellum is situated at the base of the forebrain and consists of two lateral hemispheres joined by a midline - vermis. The cerebellum, which contains the majority of the brain's neurons, is vital for neurological studies.

To generate cerebellum slices, begin with a decapitated mouse head. Insert scissors in the foramen magnum - the opening at the skull base. Gently incise the skull and remove it. Next, flip out the brain and cut the optic and trigeminal nerves. Release the brain into a chilled dissection medium with its dorsal side up. Separate the hindbrain from the other brain parts.

Then, cut the underlying cerebellar peduncles - the connection between the cerebellum and brain stem - to detach the cerebellum. Remove the meninges from the cerebellum. Carefully place the cerebellum on a tissue chopper platform perpendicular to the chopper blade. Now, dissect the tissue slices of appropriate thickness. Immediately, wet the slices and transfer them back into the Petri dish containing the dissection medium.

Separate and identify the slices from the vermis. Transfer the selected slices into a well-insert in a culture plate containing a suitable growth medium. Incubate the culture to maintain the structural and functional integrity of the dissected cerebellar slices for further experiments.

To begin, insert a 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. To retrieve the dorsal part of the skull, use a fine straight forceps to carefully lift the dorsal part of the skull.

Then, carefully introduce the forceps between the ventral skull and the brain. Gently flip out the brain and cut the optic and trigeminal nerves with small scissors. Next, turn the head or the dorsal part of the skull upside down just above a 60-millimeter cell culture dish containing ice-cold dissection medium to help the brain to drop by gravity.

Using fine forceps, orientate the brain with the dorsal side facing up and the ventral side lying down. Under the binocular microscope, use the fine straight forceps to immobilize the brain on the forebrain side. Then, separate the hindbrain from the rest of the brain. Cut the cerebellar peduncles underneath the cerebellum to separate the cerebellum from the rest of the hindbrain.

Once the cerebellum is isolated, use fine straight forceps to carefully tear away the meninges. Hold the cerebellum gently with the fine curved forceps and place it with the dorsal side up onto the plastic platform perpendicular to the chopper razor blade. Next, with a sterile thin end pipette tip attached to a 1-milliliter pipette, aspirate any excess of dissection medium around the cerebellum. Then, with a tissue chopper sliced 300-micrometer thick sagittal sections of the cerebellum.

Next, add a drop of dissection medium onto the sliced cerebellum gently. Then, with a wide-bore pipette tip attached to the 1-milliliter pipette, slowly aspirate the sliced cerebellum and transfer it back into the 60-millimeter cell culture dish containing ice-cold dissection medium.

Next, use two fine straight forceps to separate individual slices. Then, with a wide-bore pipette tip attached to the 1-milliliter pipette, transfer up to four selected slices from the vermis along with some dissection medium onto one culture insert of a 6-well plate. Remove any excess of dissection medium around the slices using a thin-end pipette tip.