Fitc conjugated goat anti mouse igg antibody

C-MSC or C-MSC pretreated with 50 ng/mL of IFN-γ for 24 h (C-MSCγ (50)) was dissociated using gentleMACS Dissociator (Milteny Biotech, Bergish Gladbach, Germany). The dissected samples were filtered through sterile 70-μm nylon cell strainers (BD) to obtain cell suspensions. The cells were then incubated with a mouse monoclonal anti-human CD90 IgG antibody (BD; 5E10), mouse monoclonal anti-human CD73 IgG antibody (BD; AD2), mouse monoclonal anti-human CD105 IgG antibody (Immunotools, Friesoythe, Germany; MEM-226), mouse monoclonal anti-CD34 IgG antibody (BD; 8G12), mouse monoclonal anti-CD45 IgG antibody (BD; 2D1), mouse anti-human human leukocyte antigen (HLA)-DR) IgG antibody (BD; G46-6), or mouse monoclonal anti-CD86 IgG antibody (BD; FUN-1) for 1 h at room temperature. The cells were then incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse-IgG antibody (Vector Laboratories Inc., Burlingame, CA, USA), for 30 min at room temperature. The expression profile of each molecule was determined using a FACScan flow cytometer (BD) with Cell Quest software (BD).

The tissue sections were incubated with blocking solution containing 0.3% Triton X-100, 5% bovine serum albumin, and 3% goat serum for 1 h followed by incubation with SMI32 mouse monoclonal antibody (1:200 dilution, Abcam), anti-LSD1 rabbit monoclonal antibody (1:200 dilution, Cell Signaling), anti-H3K4me2 rabbit monoclonal antibody (1:400 dilution, Cell Signaling), and ChAT antibody (BD Biosciences) overnight at 4 °C. After three washes with PBS, the cells were incubated for 1 h with FITC-conjugated goat anti-mouse IgG antibody (1:200 dilution; Vector Laboratories, Burlingame, CA) and Cy3-conjugated goat anti-rabbit IgG antibody (1:200 dilution; Jackson Laboratories). All antibodies were diluted in PBS. The slides were washed three times with PBS and mounted with fluorochrome mounting solution (Vector Laboratories). Moreover, images were taken by a spinning confocal microscopy (Olympus DSU, Tokyo, Japan), and the size of motor neuronal cell body was analyzed by AQI-X-COMBO-CWF program (Media cybernetics Inc. Bethesda, MD). Briefly, we took a series of 50 confocal layers representing fluorescence data from the ventral horn, then analyzed all layers by the software. Control experiments were performed in the absence of primary antibody.