Phospho-Smad3 (Ser423 + Ser425) Rabbit Polyclonal Antibody

Blank control (black line): HL60 (black) (The cells were fixed with 2% paraformaldehyde (10 min), then permeabilized with PBST for 30 min on room temperature), Primary Antibody (Red line): Rabbit Anti-Phopho-Smad3 (Ser423+Ser425) antibody, dilution: 1 µg/10^6 cells, Isotype Control Antibody (green line): Rabbit IgG. Secondary Antibody (red line): Goat anti-rabbit IgG-FITC, dilution: 1 µg/Test.

Blank control (Black line): HUVEC (Black). Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser423 + Ser425) antibody (orb6983), dilution: 1 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647, dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol 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 20000 events was performed.

Blank control: Hela. Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser423 + Ser425) antibody (orb6983), dilution: 1 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-AF647, dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at -20°C. 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 20000 events was performed.

Blank control: HUVEC (Black). Primary Antibody (green line): Rabbit Anti-Phospho-Jak1 antibody, dilution: 1 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-AF647, dilution: 1 µg/Test. Protocol, 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 20000 events was performed.

Paraformaldehyde-fixed, paraffin embedded (human gastric carcinoma), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse brain), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse kidney), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse skin), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat brain), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat heart), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat kidney), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat skin), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat testis), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (orb6983) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Sample: Lane 1: Cerebrum (Mouse) Lysate at 40 ug, Lane 2: Heart (Mouse) Lysate at 40 ug, Lane 3: Testis (Mouse) Lysate at 40 ug, Lane 4: Skin (Mouse) Lysate at 40 ug, Lane 5: Kidney (Mouse) Lysate at 40 ug, Lane 6: Cerebrum (Rat) Lysate at 40 ug, Lane 7: Testis (Rat) Lysate at 40 ug, Lane 8: Skin (Rat) Lysate at 40 ug, Lane 9: Kidney (Rat) Lysate at 40 ug, Lane 10: Huvec (Human) Cell Lysate at 30 ug, Lane 11: A549 (Human) Cell Lysate at 30 ug, Lane 12: Hela (Human) Cell Lysate at 30 ug, Lane 13: HT1080 (Human) Cell Lysate at 30 ug, Lane 14: A431 (Human) Cell Lysate at 30 ug, Primary: Anti-Phospho-Smad3 (Ser423 + Ser425) (orb6983) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 52 kD, Observed band size: 54 kD.

Tissue/Cell: human liver carcinoma, 4% Paraformaldehyde-fixed and paraffin-embedded, Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min, Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min, Blocking buffer (normal goat serum) at 37°C for 20 min, Incubation: Anti-Phospho-Smad3 (Ser423/425) Polyclonal Antibody, Unconjugated (orb6983) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAB staining.