Obtaining Cerebellum Slices from an Embryonic Chick Brain
Transcrição
To begin, place fertilized brown chicken eggs into an egg incubator at 38 degrees Celsius until they reach embryonic day 14. On embryonic day 14, cut an opening in the egg to expose the embryo, and decapitate the chick embryo de ovo. Place the head into a Petri dish containing ice-cold PBS.
Under a dissecting microscope, use standard forceps to make an incision behind each eye, all the way through the tissue, and removing the eyes and upper jaw. Then, make a second incision all the way through the pharynx to remove the lower jaw. Next, use standard forceps to remove the skin from the surface of the skull by peeling it away. Then, remove the frontal and parietal bones, revealing the brain.
From a ventral aspect, remove the pharyngeal cartilage and the auxiliary mesenchyme. Then, place the brain, dorsal side up and carefully remove the mesenchyme, dorsal to the hindbrain, taking care not to damage the pia. Next, make an incision all the way through the tissue between the midbrain and the hindbrain to detach the hindbrain, including the cerebellum. Make incisions all the way through the tissue at both the lateral junctions of the cerebellum and the alar plate of the hindbrain. Remove the entire cerebellum, taking care to maintain the integrity of the pia throughout the dissection.
Finally, remove the forming choroid plexus and move the dissected cerebellum into ice-cold HBSS. Transfer the whole cerebellum to the sterile platform of a tissue chopper, using a spatula or a 3-milliliter Pasteur pipette with the tip cut away to widen the aperture. Remove any excess liquid using a pipette.
Set the cutting speed of the tissue chopper to 50% of its maximum value. Then, cut the cerebellum in the required orientation at a thickness of 300 micrometers. Using a 3-milliliter Pasteur pipette, cover the sliced cerebellum in cold HBSS.
Then, transfer the HBSS and the slices to a 60-millimeter Petri dish containing 20 milliliters of ice-cold HBSS, using the 3-milliliter Pasteur pipette with the cut tip. Place the Petri dish under a dissecting microscope, illuminated with a fiber optic light source. Then, use a watchmaker forceps to separate individual slices of brain tissue.
