Smi 32 antibody

Ten female mice with EAE from a second cohort of animals were used for histological analysis. Peripheral blood was also collected from all the mice at the onset of the clinical signs and all the animals were sacrificed at the peak of the clinical course, when they were perfused transcardially with 4% PFA. The spinal cord of the mice was dissected out and post-fixed for 4 h at RT in the same fixative. After immersion in 30% (w/v) sucrose in PB for 12 h, coronal cryostat sections (20 μm thick: Leica) were thaw-mounted on Superfrost® Plus slides.
The same EC staining for myelin visualization was carried out as that used for the histopathology of human samples with the following modifications: the tissue was stained in 0.5% EC for 30 min, and differentiated in 5% iron alum and borax-ferricyanide for 10 and 5 min, respectively.
Axonal damage was analyzed by staining the non-phosphorylated form of the neurofilament protein (SMI-32) in spinal cord sections from mice with EAE at the peak. Immunohistochemistry was performed by incubating the sections overnight at 4 °C with SMI-32 antibody (1:200, Covance). After rinsing, the sections were then incubated for 1 h at RT with the corresponding fluorescent secondary antibody (1:1000; Invitrogen). The cell nuclei were then stained with Hoechst 33342 (10 µg/ml: Sigma-Aldrich), and the sections were mounted in Fluoromount-G (Southern Biotech).