VALIDATION IMAGES
Sample:
Lane 1: Mouse Lung tissue lysates
Lane 2: Mouse Placenta tissue lysates
Lane 3: Mouse Uterus tissue lysates
Lane 4: Mouse Spleen tissue lysates
Lane 5: Mouse Stomach tissue lysates
Lane 6: Rat Lung tissue lysates
Lane 7: Rat Placenta tissue lysates
Lane 8: Rat Spleen tissue lysates
Lane 9: Human HepG2 cell lysates
Lane 10: Human Panc-1 cell lysates
Primary:
Anti-Annexin V (bs-0450R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 36 kD
Observed band size: 36 kD
Sample:
NIH/3T3(Mouse) Cell Lysate at 30 ug
Lung (Mouse) Lysate at 40 ug
Primary: Anti-Annexin V (bs-0450R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 36 kD
Observed band size: 35 kD
Sample:
PANC-1(Human) Cell Lysate at 30 ug
Primary: Anti-Annexin V (bs-0450R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 36 kD
Observed band size: 35 kD
Sample: Lung (Mouse) Lysate at 40 ug
Primary: Anti- Annexin V (bs-0450R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 36 kD
Observed band size: 36 kD
Tissue/cell: rat lung 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-Annexin V Polyclonal Antibody, Unconjugated(bs-0450R) 1:500, overnight at 4⒉C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Tissue/cell: human Neurological glioblastoma; 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-Annexin V Polyclonal Antibody, Unconjugated(bs-0450R) 1:500, overnight at 4⒉C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Tissue/cell: rat testis 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-Annexin V Polyclonal Antibody, Unconjugated(bs-0450R) 1:500, overnight at 4⒉C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
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-Annexin V Polyclonal Antibody, Unconjugated(bs-0450R) 1:500, overnight at 4⒉C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
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-Annexin V Polyclonal Antibody, Unconjugated(bs-0450R) 1:500, overnight at 4⒉C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining
Blank control: RSC96 (blue).
Primary Antibody:Rabbit Anti-Annexin V antibody(bs-0450R), 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) , then permeabilized with 90% ice-cold methanol for 30 min on ice. Antibody (bs-0450R, 1μg /1x10^6 cells) 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 of bs-0450R at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.