Injection directly into spinal cord tissue bypasses innate biological barriers, providing access to cell bodies or synapses where molecules can be incorporated. The experimental process of stereotactic injection into spinal cord of the mouse is as follows. Prepping mice for surgery
● Before surgery, mouse is anesthetized by intraperitoneal injection of 2 % sodium pentobarbital (50 mg/kg). Wait until the mouse becomes motionless (~5 min).
● Remove the fur on the mouse’s back using electric shaver for small animals, then sterilize the skin surface on the back with iodophor and 75 % ethanol in turn. Care for the eyes of mice with aureomycin ointment.
● Make an incision on the mouse’s back (about the highest point of the hunched back in the prone position) and carefully scrap the muscle layer away to expose the vertebra T13 to L1 with disposable scalpel.
● Mount the spine to the stereotaxic apparatus with ear bars to ensure the stability. Use the overcoil tweezers to further clear muscle and spinal dural away from the intervertebral space, thereby exposing a white spinal cord.
Equipment setup
● Fill the injection electrode tube with mineral oil and fix it on the micro-injection pump. Use the micro syringe to suck 150 nl of air, then suck an appropriate amount (400-600 nl) of virus.
● Lower the injection electrode towards the spinal cord, and locate the z-axis of the apparatus "0" when just touching the surface of the spinal cord. Attach the syringe to the spinal cord when the z-axis is at a 45° angle and the entry distance is 430 μm (equivalent to a vertical depth of 300 μm).
● Start injecting at a rate of 40 nl/min after needle retaining for 2 minutes. When half of the virus is injected, stop to retain the needle for 2 minutes and retract the needle until the needle is out of the z-axis depth of 210 μm (equivalent to the vertical depth of 150 μm). Then inject all remaining viruses after 2-minute retaining needle.
Post-injection care
● After the injection is finished, wait about 3 minutes to ensure that any residual virus has been absorbed. Note that keep the skin surface moist with  normal saline during the injection.
● Suture the incision, then sterilize the skin surface with iodophor again and pull the skin together using tissue adhesive.
● Place the mouse back in its cage and keep the temperature with an electric heater until it wakes up.
● In this experiment, the virus injection site is the anterior and posterior intervertebral foramen of the right vertebra L1, to the corresponding region of the spinal cord segment L3-L4. Other experiments, such as behavioral experiments, should be carried out after 1 weeks of post-surgery. Model building, behavioral tests, immunohistochemical tests and other operations should perform after 2-3 weeks of virus expression.
NOTE: The methods of virus injection in various site are similar, just alter the injection dosage according to the characteristics and titer of different viruses. It is recommended to conduct pre-experiment before using each virus.

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