Intracranial inoculation of mice

　　1. Make the trocar:

　　1.1 Use a self-sealing strip to wrap the needle of the micro-injector so that the length of the exposed needle tip is 3 mm.

　　2. Instrument disinfection:

　　2.1 Refer to the "Maintenance and Disinfection of Surgical Instruments" SOP.

　　3. Injection site:

　　3.1 The injection site is located in the white matter area of the right frontal lobe of the mouse, 1 mm from the coronal suture and 2.5 mm from the sagittal suture.

　　4. Vaccination method:

　　4.1. During the entire operation, cells are kept on ice;

　　4.2. Inject the anesthetic, detect the degree of anesthesia of the animal using the hind limb reflex, and gently pinch the hind limb of the mouse with your hands. When the mouse does not respond, it means it has entered a state of deep anesthesia. At this time, start the experiment;

　　4.3. Place the mouse flat on the experimental table in a prone position with its head as horizontal as possible. Disinfect the head skin with iodophor and use 75% alcohol to remove iodine. Use surgical scissors to cut the skin 0.3cm downward along the sagittal suture about 0.6cm above the middle of both eyes to expose the skull. Use a sterilized dry cotton swab to bluntly peel off the connective tissue until the coronal and sagittal sutures are clearly visible. . Fix the head of the mouse with the index finger and thumb of your left hand, hold the needle in your right hand and slowly insert it into the injection site vertically and horizontally. The needle tip enters the skull about 2 mm until it reaches the white matter caudate nucleus of the frontal lobe of the brain, and slowly pushes the cells into the skull (2 μL per The injection time should be more than 2 minutes), and the needle tip cannot be moved during the process to avoid damaging the brain tissue. Suture the skin layer intermittently with a No. 6-0 suture needle, place the mouse on the heating pad, and return it to the cage after the mouse wakes up.

　　5. Precautions

　　5.1 During the operation, always pay attention to the breathing condition of the mouse, and do not fix the left hand too tightly to avoid compressing the trachea and causing suffocation;

　　5.2 After the injection, stay for 30 seconds, slowly pull out the needle, wipe away the spilled brain fluid, and suture the skin;

　　5.3 During the entire operation, cells are kept on ice;

　　5.4 The entire surgical process must be performed aseptically;

　　5.5 The skin should be sutured with interrupted suturing;

　　5.6 The injection volume shall not exceed 4 μL.