Rabbit anti map2 antibody

Nafamostat mesilate (C₁₉H₁₇N₅O₂.2CH₄0₃S, 6-amino-2-naphthyl p-guanidinobenzoate dimethanesulfonate), a serine protease inhibitor, was obtained from NANJING D&R PHARMACEUTICAL COMPANY (China). Argatroban (C23H36N6O5S•H2O, 1-[5-[(aminoiminomethyl)amino]-1-oxo-2 -[[(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl]amino]pentyl]-4-methyl-2-piperidinecarboxylic acid) was purchased from Enzo Life Sciences (BML-PI146, USA). Fluorescein isothiocyanate (FITC) dextran was purchased from Sigma-Aldrich (USA), goat anti-thrombin antibody was purchased from Santa Cruz Biotechnology (USA), rabbit anti-MAP-2 antibody was purchased from Chemicon (USA), β-actin monoclonal antibody was purchased from Sigma-Aldrich (USA), Alexa Fluo-488 conjugated goat anti-rabbit IgG antibody and Alexa Fluo-594 conjugated donkey anti-goat IgG antibody were purchased from Invitrogen (CA). RIPA buffer and MicroBCA kit were purchased from Beyotime (China). Protease inhibitor cocktail was purchased from Roche (Indianapolis, IN, USA). Thrombin substrate Benzoyl-Phe-Val-Arg-AMC·HCl and Prolyl Endopeptidase Inhibitor II were purchased from Merck (Germany).

MUSE-NPCs, 200 μL, at 1 × 10⁵/mL, were incubated in polylysine-coated 48-well plates. Each well contained 100 μL neural induction medium and 0, 30, 60, 90 or 120 ng/mL PDGF-BB. After 1, 3, 5 and 7 days of culture, cell growth was observed using the microscope. Cell proliferation rate was determined with the CCK-8 kit (Beyotime). Cells were cultured with 120 ng/mL PDGF-BB for 7 days, subjected to immunocytochemical staining using rabbit anti-MAP-2 antibody (1:1000; Millipore, CA, USA) and FITC-labeled goat anti-rabbit IgG antibody (1:500; Sigma-Aldrich), and observed by fluorescence microscopy (TCS SP8; Leica, Wetzlar, Germany).
MUSE-NPCs, 200 μL, at 1 × 10⁴/mL, were seeded in the upper chamber of a 6.5 mm Transwell (pore size: 8.0 µm; Corning, NY, USA). Neural induction medium, 500 μL, containing 0 or 120 ng/mL PDGF-BB, was added to the lower chamber. After 12 hours of culture, the membrane was stained with crystal violet. Four fields were randomly selected, and cells traversing to the lower chamber were quantified and analyzed with the light microscope (IX70; Olympus). For another experiment, 200 μL of a MUSE-NPC cell suspension, 1 × 10⁴/mL, was seeded into culture wells containing 10 mg of partition-type tubular scaffold and PDGF-MSs along with 120 ng/mL PDGF-BB and cultured for 1 month.