Cnpase

Frozen sections were stained using the CryoMyelin Kit (Hitobiotech), which allows sensitive localization and visualization of myelin fibers. The mean intensity of myelin staining in the region of interest was quantified using a Nikon Eclipse-Ti inverted microscope and NIS-elements software. The lesion area was studied in detail using immunofluorescence and western blot analyses. The various white matter-associated antibodies used for immunofluorescence and western blot analyses were CNPase (1:500, Sigma-Aldrich), NF (1:100, Dako) and NogoA (1:100, Abcam), followed by goat anti-mouse and anti-rabbit Alexa Fluor 488 (1:750, Invitrogen). For the western blot analysis, the units were normalized based on Β-actin (1:400, Sigma-Aldrich). To quantify the expression of white matter-associated markers, the mean fluorescence intensity was evaluated using a 40X objective lens (4 animals in each group, 5 sections in each animal per group, 10 different microscope fields). The experiments, images and quantification of the samples were performed by blinded observers, using the same microscope configurations, to eliminate bias due to background normalization.

The EVs were labeled with DiI (Celltracker CM-DiI, Invitrogen) prior to administration. Cryosections (40 μm thick) of lung, liver, spleen and brain were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI), and the biodistribution was analyzed by immunofluorescence staining at 24 hours.
In order to characterize which cell type contains the EVs administered to the brain, we co-stained with DiI-labeled EVs and VEGF (1:500, Millipore), NeuN (1:100, Millipore), CNP-ase (1:500, Sigma-Aldrich) and Iba-1 (1:1000, Millipore), followed by the goat anti-mouse and anti-rabbit Alexa Fluor 488 antibody (1:750, Invitrogen). Images were acquired as a confocal maximum projection using a Leica TCS-SPE confocal microscope (Leica) and the number of double-positive cells was counted in a minimum of 6 different microscopic fields using a 40× objective lens and Image-ProPlus 4.1 software.

Mouse monoclonal antibody IgM O4 was purified from hybridoma supernatant [35 (link)]. Commercial antibodies were CNPase (Sigma, C5922), MBP (Millipore, AB980), GFAP (Millipore, AB5804) and β-III tubulin (R&D MAB1195).

Cells or a 3-cm length section of injured spinal cord was lysed in lysis buffer on ice. Following electrophoresis, the collected proteins were transferred to a PVDF membrane and incubated overnight with primary antibodies at 4 °C (Cnpase, 1:500, Sigma, Germany; BMP2, 1:1000, Sigma, Germany; TGF-β, 1:1000, Invitrogen, USA; p-Smad2, 1:1000, Invitrogen, USA). The membrane was then incubated with the secondary antibodies (Elabscience; 1:5000 in blocking solution) at room temperature for 1 h. The blots were then visualized using the SuperSignal West Pico enhanced chemiluminescence reagent (Thermo Scientific) and quantified using ImageJ software. The full-length original gels are included in Additional file 2: Figure S2.

NSPCs were seeded at 5.0 × 10⁴ cells per well in 8-well poly-l-ornithine- and fibronectin-coated Lab-Tek II Chamber Slides (Nalge Nunc). They were incubated for 6 days with or without 20 μM PJ34 and the medium was changed every other day. Conversely, cells for the positive controls of neurons, astrocytes, and oligodendrocytes were incubated for 1 day in neural stem cell medium and then the medium was changed to 1× DMEM/F-12 supplemented with B-27 for differentiation and incubated for 6 days. The cells were fixed in acetone/methanol for 2 min. The antibodies to detect the following antigens were used for immunocytochemistry: nestin (sc-20978, 1:25; Santa Cruz Biotechnology or MAB353, 1:200; Chemicon), beta-III tubulin (MAB1195, 1:100; R&D Systems), GFAP (Z0334, 1:500; Dako Cytomation), CNPase (MAB326, 1:200; Chemicon), p21 (sc-53870, 1:100; Santa Cruz Biotechnology), p53 (2524, 1:100; Cell Signaling), and phospho-p53 (Ser18) (9284, 1:50; Cell Signaling). Alexa Fluor dye-conjugated secondary antibodies of donkey anti-mouse IgG-Alexa Fluor 488 (A21202, 1:500; Molecular Probes) and goat anti-rabbit IgG-Alexa Fluor 568 (A11036, 1:500; Molecular Probes) were used for detection. Nuclear staining was performed using 1 nM 4′, 6-diamidino-2-phenylindole (17514; ABD Bioquest). Cellular fluorescence images were acquired using a confocal laser scanning microscope (LSM 510; Carl Zeiss).