Preparation of CNS Tissue for Isolating Cells in EAE Mice

Begin by placing the mouse in supine position and fixing the limbs with needles. Apply 75%ethanol on the front body of the animal. Make a longitudinal section through the skin and fascia on the abdomen and thorax.

Cut the ribs parasternal and fold up the thorax to gain free access to the heart. Fix the thorax folded upwards with needles. Open the right atrium using scissors.

Using a cannula, apply 20 milliliters of one-time PBS into the left ventricle to flush out the blood through the incised right atrium. Make a longitudinal section by cutting the skin on top of the head of the mouse to expose the skull and shift the skin around the head using forceps. Incise the skull with the help of a scissor along the sagittal suture.

Insert the tip of the scissors along the incision line to crack open the calotte. Remove the remaining parts of the calotte with forceps so that the brain is fully exposed. Remove the brain carefully and place it into a murine brain matrix.

Cut the brain into one millimeter thick sagittal slices by using a razor blade. With the help of scissors, cut the vertebral column just above the iliac crest. Insert a 20 gauge needle attached to a 20 milliliter syringe containing one-time PBS into the spinal canal.

Flush the spinal cord out of the spinal canal from caudal to cranial. Cut the spinal cord into 0.5 centimeter long segments using a scalpel. Store the CNS cell suspension consisting of the brain and corresponding spinal cord in a Petri dish containing three milliliters of cold DPBS.