Adhesive coated slides

The animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and then cardio-perfused with 4% PFA solution. The 8 mm-long spinal cord tissues, both 4.0 mm rostral and caudal to the lesion site, were dissected out and postfixed in the same fixative overnight at 4 °C. The tissues were then soaked in cold PBS containing 20% sucrose overnight at 4 °C, and subsequently frozen in embedding compound (Sakura Finetek, Tokyo, Japan). Sagittal serial sections 25-μm thick were prepared with a cryostat (model CM 1850; Leica, Nussloch, Germany), attached to adhesive-coated slides (Matsunami Glass), and dried before being used for immunofluorescence studies.

Mice were transcardially perfused with PBS followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The spinal cords were dissected, postfixed in the same fixatives, and immersed overnight in PBS containing 30% sucrose, following which they were embedded in Tissue-Tek OCT and frozen at − 80 °C until use. The sections were cut on a cryostat (20 µm-thickness) and mounted on Matsunami adhesive-coated slides (Matsunami, Osaka, Japan). Cryostat sections were incubated with blocking solution containing 5% bovine serum albumin and 0.1–0.3% Triton X-100 in PBS for 1 h at room temperature, followed by overnight incubation with the indicated primary antibodies at 4 °C. Immunoreactivity was visualized using fluorescence-conjugated secondary antibodies. The samples were then coverslipped with mounting medium (Dako) and examined under a fluorescence microscope (Olympus BX53, DP71).