VALIDATION IMAGES
Sample:
Lane 1: MCF-7 (Human) Cell Lysate at 30 ug
Lane 2: HepG2 (Human) Cell Lysate at 30 ug
Lane 3: Molt-4 (Human) Cell Lysate at 30 ug
Lane 4: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Primary: Anti- PCNA (bsm-33035M) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 36 kD
Observed band size: 35 kD
Paraformaldehyde-fixed, paraffin embedded (Human colon 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by conjugation to the secondary antibody (labeled with HRP)and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human stomach 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Mouse) (sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Rat liver); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0024) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Mouse testis); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0024) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human rectal 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human tonsil); 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse spleen); 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat testis); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse 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; Incubation with (PCNA(Nuclear Loading Control) ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human colon); 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; Incubation with (PCNA(Nuclear Loading Control) ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse lymphoid); 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human cervical 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Descending colon cancer); 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human stomach 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Mouse) (sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human liver cancer); 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 (PCNA) Polyclonal Antibody, Unconjugated (bsm-33035M) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Mouse) (sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human esophageal cancer); 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 (PCNA(Nuclear Loading Control) ) Monoclonal Antibody, Unconjugated (bsm-33035M ) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse testis); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat testis); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human tonsil); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human cervical 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat lymphoid); 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 (PCNA) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse colon); 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse testis); 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat colon); 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat testis); 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat liver); 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Mouse small intestine); 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand 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 (PCNA ) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human cervical 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human gastric 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat spleen); 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 (PCNA(Nuclear Loading Control)) Monoclonal Antibody, Unconjugated (bsm-33035M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.
Blank control:Jurkat.
Primary Antibody (green line): Mouse Anti-PCNA antibody (bsm-33035M)
Dilution: 1:50;
Secondary Antibody : Goat anti-mouse 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.