Isolation Protocol of Subependymal Zone of Mice
GUIDELINE
The subependymal zone is located near the striatum in the wall of the lateral ventricle and produces olfactory bulb interneurons.
METHODS
- Mice are killed by cervical dislocation. Shears are used to cut off the head and 70% ethanol is sprayed liberally to increase sterile conditions and minimize airborne diffusion of the fur during dissection.
- Use small scissors to remove the skin and expose the skull surface. The bone is cut lengthwise along sagittal sutures without damaging the lower brain tissue.
- The skull is peeled off with curved or straight tweezers, and the brain is scooped out with a scraper and placed in a slab with cold DPBS.
- The olfactory bulb is first removed, then the midbrain is cut vertically (using the midbrain cap as a guide) to obtain a 4-5 mm thick section containing the lateral ventricles.
- Discard the rest of the tissue and separate the hemispheres one at a time.
- Tissue is opened along the corpus callosal line that separates the hippocampus, septum, and diencephalon from the cortex. This will expose the subependymal area.
- Trim the subependymal zone (SEZ) and remove all surrounding tissue. The surface of the subependymal zone is dissected carefully to obtain the thinnest tissue sections possibly. The subependymal zone is then distinguished from the parenchyma.
- Two anatomical subependymal zones of each brain are transferred into p6 holes with sterile cold DPBS and cut into 3-4 small pieces with a small scalpel.
NOTES
- Clean work surfaces thoroughly with 70% ethanol to avoid contamination. The anatomical instrument is sterilized by placing it in a glass beaker containing 70% ethanol.
- p6 well culture plastic plates are filled with cold sterile DPBS, and it is used to place and clean brains of mice before dissection. Place the plate on the ice.
- Based on the number of mice and the experience of the researchers, estimate the time required to complete the dissection. Be quick!
