Sample:Raji Cell Lysate at 40 ug
Primary: Anti-CCR7 (JP122289) at 1:300 dilution;
Secondary: HRP conjugated Goat-Anti-Rabbit IgG at 1: 5000 dilution;
Predicted band size : 42 kD
Observed band size : 42 kD

Blank control (Black line): Mouse spleen(Black). Primary antibody: Rabbit Anti-CD4 antibody Dilution: 3μg /10^6 cells; Isotype Control antibody: Rabbit IgG-PE.Secondary antibody: Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
ProtocolThe cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol 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.

Blank control: Raji(blue).
Primary Antibody:Rabbit Anti-CCR7 antibody, Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG ,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 were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 10% 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.