Mouse anti cnpase

Mouse anti-CNPase, rabbit anti-Hes1, and rabbit anti-Hes5 antibodies were purchased from Abcam Corporation (Cambridge, UK). Anti-MBP antibodys were obtained from Millipore Corporation (Billerica, MA) for immune staining or ABcam (ab62631) for Western blot, and immobilon Western chemiluminescent HRP substrate were obtained from Millipore Corporation. Mouse anti-β-actin monoclonal antibody was purchased from CWBIO Corporation (Beijing, China). HRP-linked anti-rabbit and anti-mouse IgG antibodies were from Cell Signaling Corporation (Beverly, MA). Alexa Fluor 594 Donkey anti-rat IgG was from Invitrogen Corporation (San Diego, CA), and primers of Notch1, Notch2, Notch4, Hes1, and Hes5 were synthesized by TaKaRa Corporation (Seta, Japan). PrimeScript RT reagent Kit with gDNA Eraser, SYBR Premix Ex Taq (TliRNaseH Plus), and EASY Dilution were also from TaKaRa Corporation. Cuprizone (bis-cyclohexanoneoxalydihydrazone, CPZ) was purchased from Sigma Corporation (Ronkonkoma, NY). Quetiapine was a generous gift from Professor Li Xinmin, University of Manitoba, Canada. Halothane was obtained from Halocarbon Laboratories (Kinderkamack, NY). Biodentine dental cement was obtained from Septodont Corporation (Saint Maur des Fossés, France). DMSO was obtained from Sigma Corporation, and MW167, a γ-secretase II inhibitor, was purchased from Calbiochem Corporation (La Jolla, CA).

Animals were euthanized by overdose of pentobarbital. After transcardial perfusion with PBS and 4% paraformaldehyde, spinal cords were excised under a dissection microscope and post-fixed in paraformaldehyde at 4 °C for another 8 h, followed by cryoprotection using 30% sucrose. The spinal cord tissue including lesion area was embedded in OCT compound and sliced horizontally to produce 15 µm frozen sections using a cryostat microtome (Leica). Fluorescence immunohistochemistry was performed using following primary antibodies: rabbit anti-glia fibrillary acid protein (GFAP, 1:1,000, Dako), rat anti-CD45 (ebioscience, 1:1,000), rat anti-CD11b (ebioscience, 1:500), rabbit anti-IBA1 (WAKO, 1:500), rabbit anti-SNAP25 (Synaptic Systems GmbH, 1:1,000), purified anti-Neurofilament Marker (pan axonal, cocktail, SMI-312) (Biolegend, 1:1,000), mouse anti-CNPase (Abcam, 1:1,000), rabbit anti-PLP1 (Abcam, 1:1,000), fluorescence labeled Lectin (Vector, 1:20), chicken anti-MAP2 (Abcam, 1:1,000), rat anti-NeuN (Abcam, 1:1,000).

Equal amounts of protein (7.5–15 µg) from brain membranes were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were blocked in 3% dry milk powder dissolved in Tris-HCl pH 7.4 buffered saline containing 0.3% Triton X-100 for at least 1 hr and incubated with primary antibody overnight at 4°C. Primary antibodies included mouse anti-βIII tubulin (1:20,000; Promega, WI), rabbit anti-MAG (1:1000; Winters et al., 2011 (link)), rat anti-MBP (1:1000; Millipore), mouse anti-CNPase (1:1000, Abcam), anti-PLP (1:1000, Abcam), and mouse anti-Fig4 (1:200, NeuroMab, CA). Primary antibodies were detected using either horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000–15000; Millipore Bioscience Research Reagents) or Alexa-conjugated secondary antibodies (1:20,000, Molecular Probes). The Licor C-DiGit and Odyssey imaging systems and software were used for visualization and quantification of protein bands (Licor, NE).