VALIDATION IMAGES
Hela (Positive) and Molt-4 (Negative control) cells (black) were fixed with 4% PFA for 10min at room temperature, permeabilized with 90% ice-cold methanol for 20 min at -20℃, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with FoxO1 (Ser256) Antibody (bs-3142R) at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2% BSA in PBS, followed by secondary antibody(blue) incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
HepG2 cells were fixed with 4% PFA for 10min at room temperature,permeabilized with 90% ice-cold methanol for 20 min at -20℃, and incubated in 5% BSA blocking buffer for 30 min at room temperature. Cells were then stained with Phospho-FoxO1 (Ser256) Polyclonal Antibody(bs-3142R)at 1:100 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. Acquisitions of 20,000 events were performed. Cells stained with primary antibody (green), and isotype control (orange).
Paraformaldehyde-fixed, paraffin embedded Rat lung; 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Mouse 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Rat brain; 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Rat placenta; 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, DAB staining.
Paraformaldehyde-fixed, paraffin embedded Mouse brain; 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 FoxO1 (Ser256) Polyclonal Antibody, Unconjugated (bs-3142R) at 1:200 overnight at 4°C, DAB staining.