Ab 11779

Cells grown on glass coverslips were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT), and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Fixed cells were blocked with 5% BSA in PBS for 30 min, incubated overnight at 4°C with primary Ab, washed, incubated 1 h at RT with secondary Ab, and counterstained with DAPI (Pharmingen). Coverslips were mounted with glycerol mounting medium (Dako) and sealed with clear nail polish. Fluorescence images were obtained by confocal immunofluorescence microscopy (model TCS SP8; Leica).
Primary Abs used for immunofluorescence staining were anti-GD2 (mouse, Clone 14.G2a, #554272, BD Biosciences), anti-GD3 (mouse, clone R24, ab 11779, Abcam), anti-EGFR (rabbit, ab32562; Abcam), and anti-c-Met (rabbit, sc-10, Santa Cruz). Secondary Abs used were Alexa555-conjugated donkey anti-mouse IgG and donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa488-conjugated donkey anti-rabbit IgG and donkey anti-mouse IgG (Invitrogen).

Cells were detached with trypsin, and single cells were washed and resuspended in PBS containing 2% FBS. Cell suspensions were incubated with optimal concentrations of anti-GD2 mAb (Clone 14.G2a, #554272, BD Biosciences) or anti-GD3 mAb (R24 clone, ab 11779, Abcam) for 20 min at 4°C. Cells were washed and then incubated at 4 °C with fluorescence-labeled secondary Ab for 20 min. Control experiments were performed using appropriate isotype controls, or secondary Ab alone. Cells were subjected to flow cytometric analysis using a SA3800 spectral cell analyzer and FlowJo software program.