Anti cnpase

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).

For immunohistochemical, immunocytochemical, and Western blot procedures please reference the Supplemental Experimental Procedures. Antibodies used for immunohistochemistry were anti-GFP (Abcam, 1:500), anti-Brdu (Accurate,1:200, 30 min 2N HCl followed by 15 min 0.1M Boric Acid brain section pre-treatment), anti-NG2 (Millipore, 1:500), anti-GFAP (Sigma, 1:500), anti-ET-1 (Abbiotec, 1:200), anti-CD31 (BD Biosciences, 1:500), anti-Jagged-1 (Iowa Hybridoma Bank, 1:200), anti-IBA1 (Wako, 1:500), anti-MAG (Santa Cruz, 1:200), anti-MBP (Covance (SMI-99p), 1:1000), anti-Hes1 (Millipore, 1:1000), anti-CD11b/MAC1 (ABD Serotec, 1:400), anti-Olig2 (Millipore, 1:500), and anti-APC (Ab-7) (CC-1) (Calbiochem, 1:500). Antibodies used for immunocytochemistry were anti-GFP (Abcam, 1:500), anti-O1 (R&D systems, 1:500), anti-GFAP (Sigma, 1:500), and anti-NG2 (Millipore, 1:500). Antibodies used for Western Blot analysis include anti-MBP (Covance (SMI-99p), 1:5000), anti-MAG (Santa Cruz (sc-15324), 1:200), anti-CNPase (Covance, 1:500), anti-Jagged1 (Santa Cruz (sc-135955), 1:200), anti-β-actin(C4) (Millipore, 1:5000), anti-GFAP (Sigma, 1:5000), and anti-NICD (Iowa Hybridoma Bank C17.9C6, 1:1000).