VALIDATION IMAGES
Sample:
Lane 1: Large intestine (Mouse) Lysate at 40 ug
Lane 2: Stomach (Mouse) Lysate at 40 ug
Lane 3: Uterus (Mouse) Lysate at 40 ug
Lane 4: Kidney (Mouse) Lysate at 40 ug
Lane 5: Small intestine (Mouse) Lysate at 40 ug
Lane 6: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Lane 7: Stomach (Rat) Lysate at 40 ug
Lane 8: Uterus (Rat) Lysate at 40 ug
Lane 9: A549 (Human) Cell Lysate at 30 ug
Lane 10: Hela (Human) Cell Lysate at 30 ug
Lane 11: A431 (Human) Cell Lysate at 30 ug
Lane 12: HepG2 (Human) Cell Lysate at 30 ug
Primary: Anti-alpha smooth muscle Actin (bsm-33187M) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD
Paraformaldehyde-fixed, paraffin embedded (Human uterus); 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (bsm-33187M) 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 uterus); 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (bsm-33187M) 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 uterus); 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (bsm-33187M) 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 stomach); 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (bsm-33187M) 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 stomach); 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (bsm-33187M) 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 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (bsm-33187M) 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 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (ascites of bsm-33187M 3F9) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Mouse) (sp-0024) instructions and 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 (alpha smooth muscle Actin) Monoclonal Antibody, Unconjugated (ascites of bsm-33187M 3F9) at 1:2000 overnight at 4°C, followed by operating according to SP Kit(Mouse) (sp-0024) instructions and DAB staining.
Paraformaldehyde-fixed, paraffin embedded Human Breast Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded Human Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded Human Stomach; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded Mouse Stomach; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded Rat Stomach; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded Human Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Paraformaldehyde-fixed, paraffin embedded Mouse Colon; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with alpha smooth muscle Actin Monoclonal Antibody, Unconjugated (bsm-33187M) at 1:200 overnight at 4°C. Followed by conjugated Goat Anti-Mouse IgG antibody (green, bs-0296G-BF488), DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control:NIH/3T3.
Primary Antibody (green line): Mouse Anti-alpha smooth muscle Actin antibody (bsm-33187M)
Dilution: 1ug/Test;
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.