Anti mag

Western Blot Analysis was performed on lysates from ipsilateral brain samples in order to confirm our proteomics results. Equivalent amounts of protein from each sample were subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis using 4–12% Bis–Tris precast gels (Invitrogen, California, USA) under reducing and non reducing conditions (1h, RT) and electro-blotted onto a nitrocellulose membrane (18h, Overnight, Bio Rad). Following a blocking step (0.1% Tween-20/5% nonfat milk in PBS, 1h, RT) membranes were incubated with primary antibodies overnight (12–14 h, 4°C) with gentle agitation. The following primary antibodies were used (1:1000): Anti-MBP (Millipore), Anti-MAG (AbCam), Anti-Beta Actin (Cell Signaling). Membranes were washed, incubated with secondary antibody (RT, 1h, Cell Signaling) and developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).

Specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Slices 4μm thick were stained with hematoxylin and eosin, Luxol fast blue/periodic acid–Schiff, and Bielschowsky silver impregnation. Immunohistochemical staining was performed using an avidin–biotin technique. The following primary antibodies were used for classification of lesions: anti–myelin basic protein (anti-MBP; Dako, Glostrup, Denmark), anti–proteolipid protein (anti-PLP; Biozol Diagnostica, Eching, Germany), anti–2′,3′-cyclic nucleotide 3′-phosphodiesterase (anti-CNPase; Covance, Princeton, NJ), anti–myelin oligodendrocyte glycoprotein (anti-MOG; Abcam, Cambridge, UK), anti–myelin-associated glycoprotein (anti-MAG, Abcam), anti-KiM1P (macrophages, Dr Radzun, University of Göttingen, Germany), anti-CD3 (T cells, Dako), and anti-CD8 (cytotoxic T cells, Dako). To analyze the acute axonal injury, we used an antibody against the beta-amyloid precursor protein (anti-APP; Chemicon, Millipore, MN).