VALIDATION IMAGES
Sample:
Lane 1: A549 (Human) Cell Lysate at 30 ug
Lane 2: MCF-7 (Human) Cell Lysate at 30 ug
Lane 3: HepG2 (Human) Cell Lysate at 30 ug
Primary: Anti-Cytokeratin 19 (bs-2190R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Paraformaldehyde-fixed, paraffin embedded (Human stomach carcinoma); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (Cytokeratin 19) Polyclonal Antibody, Unconjugated (bs-2190R) at 1:400 overnight at 4℃, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human cervical carcinoma); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (Cytokeratin 19) Polyclonal Antibody, Unconjugated (bs-2190R) at 1:400 overnight at 4℃, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human liver carcinoma); Antigen retrieval by microwave in sodium citrate buffer (pH6.0) ; Block endogenous peroxidase by 3% hydrogen peroxide for 30 minutes; Blocking buffer (3% BSA) at RT for 30min; Antibody incubation with (Cytokeratin 19) Polyclonal Antibody, Unconjugated (bs-2190R) at 1:400 overnight at 4℃, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.
Blank control:MCF-7.
Primary Antibody (green line): Rabbit Anti-Cytokeratin 19 antibody (bs-2190R)
Dilution: 1ug/Test;
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 0.5ug/Test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃.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:A549.
Primary Antibody (green line): Rabbit Anti-Cytokeratin 19 antibody (bs-2190R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
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.
Blank control (blue line): Hela (fixed with 70% methanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃).
Primary Antibody (green line): Rabbit Anti-Cytokeratin 19 antibody (bs-2190R),Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE,Dilution: 1μg /test.
Blank control(blue): MCF7(fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice). Primary Antibody: Rabbit Anti-Cytokeratin 19/FITC Conjugated antibody (bs-2190R /FITC), Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG/FITC(orange) ,used under the same conditions.
