Sample:
MCF-7(Human) Cell Lysate at 40 ug
Primary: Anti-CD34 (bs-0646R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 39 kD
Observed band size: 115 kD

Sample:
Huvec(Human) Cell Lysate at 30 ug
Molt-4(Human) Cell Lysate at 30 ug
Primary: Anti-CD34 (bs-0646R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 120 kD
Observed band size: 120 kD

Tissue/cell: human lung 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-CD34 Polyclonal Antibody, Unconjugated(bs-0646R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: mouse heart tissue; 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-CD34 Polyclonal Antibody, Unconjugated(bs-0646R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: human gastric adenocarcinoma;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-CD34 Polyclonal Antibody, Unconjugated(bs-0646R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, AF488 conjugated(bs-0295G-AF488)used at 1:200 dilution for 40 minutes at 37°C.

Blank control: Raji(blue).
Primary Antibody:Rabbit Anti- CD34 antibody(bs-0646R), 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). Antibody (bs-0646R, 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 of bs-0646R at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.

Blank control: HUVEC.
Primary Antibody (green line): Rabbit CD34 antibody (bs-0646R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody: Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
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.