WT and DKO mice were perfused with PBS and then 4% paraformaldehyde in 0.1 M phosphate buffer. The spinal cord was removed, post-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4°C, then replaced with 10% sucrose for 6 hours, 15% sucrose for 6 hours, 20% sucrose for 6 hours, and embedded in OCT compound (Sakura Finetechnical, Nagoya, Japan) and frozen in liquid nitrogen. Frozen sections were set at longitudinal orientation for the spinal cord and were cut into 7 μm-thick sections on a cryostat (CM3050S; Leica, Wetzlar, Germany) at −20°C. The sections were placed on MAS-coat grids (Matsunami Glass, Osaka, Japan), and tissue slices were stored at −80°C until used.
For immunohistochemistry, tissue slices were washed twice with PBS for 5 minutes at room temperature, blocked with 10% normal goat serum for 1 hour at room temperature, and incubated with primary antibodies for 1 hour at room temperature or overnight at 4°C. Then slices were washed 3 times with PBS for 5 minutes at room temperature, and incubated with Alexa Fluor-conjugated second antibodies for 45 minutes at room temperature. Then they were washed 3 times with PBS for 5 minutes at room temperature, and were sealed with PermaFlour™ aqueous mounting medium (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cover slips were mounted and observed by fluorescence microscopy.
For immunohistochemistry, tissue slices were washed twice with PBS for 5 minutes at room temperature, blocked with 10% normal goat serum for 1 hour at room temperature, and incubated with primary antibodies for 1 hour at room temperature or overnight at 4°C. Then slices were washed 3 times with PBS for 5 minutes at room temperature, and incubated with Alexa Fluor-conjugated second antibodies for 45 minutes at room temperature. Then they were washed 3 times with PBS for 5 minutes at room temperature, and were sealed with PermaFlour™ aqueous mounting medium (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cover slips were mounted and observed by fluorescence microscopy.