For immunohistochemistry, fresh frozen tissues were obtained from an uninjured spinal cord and from spinal cords at 1, 3, and 7 days after injury. After induction of deep anesthesia with diethyl ether, the rats were decapitated and the spinal cords were dissected out, embedded in Tissue-Tek OCT, and immediately frozen on dry ice at −80°C. A series of sagittal sections were cut at 16 μm on a cryostat and mounted on aminopropyltriethoxysilane (APS) coating Superfrost-Plus slides (Matsunami, Osaka, Japan). The sections were fixed in 4% paraformaldehyde for 1 h at 25°C, washed three times with PBS, and blocked in PBS containing 5% bovine serum albumin and 0.1% Triton X-100 for 1 h at 25°C. The sections were incubated with primary antibodies overnight at 4°C and washed three times with PBS, followed by incubation with fluorescein-conjugated secondary antibodies (1 : 1000; Invitrogen) for 1 h at 25°C. The anti-TuJ1 (1 : 500), monoclonal anti-MOSP (1 : 200; Chemicon, Temecula, CA, USA), monoclonal anti-glial fibrillary acidic protein (anti-GFAP) (1 : 400; Sigma–Aldrich), monoclonal anti-BMP-2/4 (1 : 50; R&D systems), polyclonal anti-BMP-RIa antibody (Abgent, San Diego, CA, USA), polyclonal anti-BMP-RII antibody (1 : 50; Abgent), monoclonal anti-CD11b antibody (BD Biosciences, San Jose, CA, USA), monoclonal anti-CSPG antibody (Sigma–Aldrich), or polyclonal anti-phospho smad1/5/8 antibody (Chemicon Int, Inc.) was used as the primary antibody. Samples were examined under a fluorescence microscope.
To assess the glial scar formation after SCI, the 10-mm-long sagittal sections of the injured spinal cords obtained from 5 Fc-treated control rats and six noggin-treated rats (as described above) were immunostained. The monoclonal anti-GFAP antibody was used as the primary antibody, and the glial scar formation was quantitatively analyzed using Scion Image software. Digital photomicroscopic images were converted into gray scale and collected using identical intensity settings with a threshold of 1-234/255 for GFAP-positive areas and a threshold of 1-248/255 for the whole area of the sagittal section. The area of GFAP-positive cells was then calculated as a percentage of the total area of each section. We calculated the estimated volume of the injured spinal cord by the same method.