Anti mag

Zebrafish spinal cord was collected 7 and 10 days after injury along with the control, uninjured cords. Tissues were prepared in extraction buffer (37.5 mM Tris, 75 mM Nacl, 0.5% Triton X-100, protease inhibitor cocktail) and then the resulting tissue lysates were subjected to either 7.5% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis, proteins were transferred onto nitrocellulose membranes and subjected to western blotting. The western blots were developed with antibody anti-CNPase (1:1000, Millipore, USA), anti- MAG (1:100, Santa Cruz Biotechnology, USA), anti- OCT4 (1:200, Millipore, USA) and anti-NG2 (1:500, Millipore, USA) followed by anti-mouse or anti-rabbit alkaline phosphatase coupled secondary antibody (1:1,000 Jackson laboratory, USA). GAPDH was used as internal loading control protein. Protein bands were visualized using NBT/BCIP as substrate. PageRuler Broad Range Protein Ladder (Thermo Scientific, USA) was used as a standard molecular weight marker.

Primary cell cultures were homogenized in RIPA buffer that was supplemented with protease cocktail inhibitors (Beyotime, Shanghai, China). Cell lysates were subjected to Western blot analysis using anti-inducible nitric oxide (iNOS; 1: 500, Cell Signaling Technology, Catalog#13120), anti-Arginase-1 (1: 1000, Santa Cruz Biotechnology, Catalog#sc-271430), anti-MBP (1: 500, MilliporeSigma, Catalog#MAB382), anti-MAG (1: 500, Santa Cruz Biotechnology, Catalog#C0217). The protein bands were analyzed and quantified using Image Lab (ODYSSEY CLX, LI-COR, America), normalizing target proteins to GAPDH (1:10000, proteintech, Catalog#10494) bands.