Blank control: HL-60. Primary Antibody (green line): Rabbit Anti-FUT4 (orb11420), Dilution: 1 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-AF647, Dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at room temperature. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
Blank control: Mouse spleen. Primary Antibody (green line): Rabbit Anti-FUT4/FITC Conjugated antibody, Dilution: 1 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG-FITC. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 0.1% PBST for 20 min at -20C. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. The cells were stained with Primary Antibody for 30 min at room temperature. Acquisition of 20000 events was performed.
Overlay histogram showing HL 60 cells stained with orb11420 (Green line). The cells were fixed with 90% methanol (5 min) and then permeabilized with 0.01M PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (orb11420, 1 µg/1x10 6 cells) for 30 min at 22C. The secondary antibody used was fluorescein isothiocyanate goat anti-rabbit IgG (H+L) (Brillant blue line) at 1/200 dilution for 30 min at 22C. Isotype control antibody was rabbit IgG (polyclonal, Orange line) (1 µg/1x10 6 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of 20000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
Paraformaldehyde-fixed, paraffin embedded (Human lung cancer), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37C for 30 min, Antibody incubation with (FUT4) Polyclonal Antibody, Unconjugated (orb11420) at 1:400 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: A549 Cell (Human) Lysate at 40 ug Primary: Anti-FUT4 (orb11420) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 58kD Observed band size: 63kD.
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