VALIDATION IMAGES
Sample:
Lane 1: Rat Liver tissue lysates
Lane 2: Rat H9C2 cell lysates
Lane 3: Human LOVO cell lysates
Lane 4: Human K562 cell lysates
Lane 5: Human HL-60 cell lysates
Primary: Anti-FCAR/CD89 (bs-6586R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 29 kDa
Observed band size: 50 kDa
Paraformaldehyde-fixed, paraffin embedded (human brain glioma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (FCAR) Polyclonal Antibody, Unconjugated (bs-6586R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human liver carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (FCAR) Polyclonal Antibody, Unconjugated (bs-6586R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human lung carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (FCAR) Polyclonal Antibody, Unconjugated (bs-6586R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (FCAR) Polyclonal Antibody, Unconjugated (bs-6586R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (FCAR) Polyclonal Antibody, Unconjugated (bs-6586R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Blank control:Jurkat.
Primary Antibody (green line): Rabbit Anti-CD89 antibody (bs-6586R)
Dilution: 2ug/Test;
Secondary Antibody (white blue line) : Goat anti-rabbit IgG-FITC
Dilution: 0.5ug/Test.
Isotype control(orange line):Normal Rabbit IgG
Protocol
The cells were 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.