The specificity of CNP/Cre has been demonstrated in previous studies (35). Histological studies and immunohistochemistry Animals underwent intracardiac perfusion under deep anesthesia Romidepsin order (pentobarbital, 50 mg/kg i.p.) with cold heparinized PBS followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). Brains were removed, postfixed in the same fixative at 4��C overnight, and cryoprotected in 30% sucrose in 0.1 M PBS for 48�C72 h. At least 3 sections (10 ��m) from each animal were stained with luxol fast blue (LFB) overnight at 56��C, and rinsed with 95% ethanol, lithium carbonate, 70% ethanol, and water. Sections were then counterstained with hematoxylin and eosin and subsequently dehydrated. LFB-stained septostriatal and rostral diencephalon sections were assessed for demyelination by 2 observers in a masked procedure, as described previously, with some modifications (36).

Demyelination scores ranged from 0 to 4 for the medial corpus callosum [0, fully myelinated; 1, mild demyelination (��1/3) in the center (see Fig. 1A; arrows); 2, moderate demyelination (��2/3); 3, no myelin in the center; and 4, demyelination extending to the arch]. For the lateral (callosal) projections, scores from 0 to 3 were used, with 0 as normal and 3 as no myelin. Figure 1. Amelioration of cupr-induced demyelination by FTY720. A) Levels of brain sections (septostriatal and rostral diencephalon) sampled for LFB staining, areas analyzed, and treatment schedule to induce demyelination. Arrows delineate medial and lateral corpus …

For immunohistochemistry, the following monoclonal antibodies (mAbs) were used: CC1 (1:50; Calbiochem, Gibbstown, NJ, USA), anti-glial fibrillary acidic protein (GFAP; 1:100), anti-phosphorylated neurofilament heavy chain (pNF-H; 1:40; Sigma), Nkx2.2 (1:100; clone 74.5A5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); anti-proliferating cell nuclear antigen (PCNA; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Polyclonal Abs included anti-myelin basic protein (MBP; 1:1000; Dako, Carpinteria, CA, USA), anti-Iba1 (1:100; Wako Chemicals, Richmond, VA, USA), anti-��-amyloid precursor protein (��-APP; 1:200; Abcam, Cambridge, MA, USA), anti-Olig2 (1:200; Millipore, Temecula, CA, USA), and anti-NG2 (1:100; Millipore). Appropriate alkaline phosphatase-labeled or peroxidase-labeled secondary antibodies (1:200) and corresponding substrates were used for detection.

For Nkx2.2, immunoreactivity was detected using the Ultravision LP detection system (LabVision; Thermo Fisher Scientific, Fremont, CA, USA). In some experiments, Alexa 488-conjugated and Alexa 594-conjugated secondary antibodies were used (1:500; Molecular Probes, Eugene, OR, USA). No staining was observed in tissue sections when primary antibodies were Dacomitinib omitted (Dako).