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Sample:
Colon carcinoma (Human) lysate at 30ug;
Heart(Rat) lysates, 30ug;
Primary: Anti-CD24 (bs-0528R) at 1:200 dilution;
Secondary: HRP conjugated Goat-Anti-Rabbit IgG(bs-0295G-HRP) at 1: 3000;
Predicted band size : 8kD
Observed band size : 35kD

Tissue/cell: human colon carcinoma; 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-CD24 Polyclonal Antibody, Unconjugated(bs-0528R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control: A549 (blue).
Primary Antibody:Rabbit Anti-CD24 antibody(bs-0528R), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions );
Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) . Primary antibody (bs-0528R, 1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.