Rabbit anti-Osteopontin Polyclonal Antibody

Tissue/cell: rat shin bone; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer (0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum) at 37℃ for 20 min; Incubation: Anti-osteopontin Polyclonal Antibody, Unconjugated 1:300, overnight at 4°C, followed by conjugation to the secondary antibody and DAB staining

Blank control: HepG2. Primary Antibody (green line): Rabbit Anti-Osteopontin antibody Dilution: 1ug/10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody : Goat anti-rabbit IgG-AF647 Dilution: 1ug/T. Protocol The cells were fixed with 4% PFA (10min 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 20,000 events was performed.

Sample: Lane 1: HepG2 (Human) Cell Lysate at 30 ug Lane 2: A549 (Human) Cell Lysate at 30 ug Lane 3: U251 (Human) Cell Lysate at 30 ug Primary: Anti-Osteopontin at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 34 kD Observed band size: 60 kD

Paraformaldehyde-fixed, paraffin embedded (Rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Osteopontin) Polyclonal Antibody, Unconjugated at 1:400 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.