*4.5. Brain Sample Preparation*

Mice were sacrificed at 24 h or 3 days after GCI using urethane (1.5 g/kg, i.p.) to deeply anesthetize them. After anesthetizing, mice were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde (PFA). Afterwards, harvested brains were post-fixed for approximately 1 h in 4% PFA. After fixation in PFA, brains were moved into a 30% sucrose solution overnight for cryoprotection. After the brain sank to the bottom of the sucrose solution, they were frozen in the freezing medium for 10 min and then cut with cryostats at 30 μm thicknesses. Brain slices were kept in storage solution until used for immunohistochemistry and immunofluorescence staining.
