VALIDATION IMAGES
Sample:
Lane 1: MCF-7 cell lysates
Primary: Anti-Phospho-Estrogen Receptor alpha (Ser118) (bsm-52154R) at 1/500 dilution
Secondary: Goat Anti-Rabbit IgG - HRP at 1:5000 dilution
Predicted band size: 66 kD
Observed band size: 66 kD
Paraformaldehyde-fixed, paraffin embedded (human breast); 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 (Phospho-Estrogen Receptor alpha (Ser118)) Polyclonal Antibody, Unconjugated (bsm-52154R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand 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 Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Endometrial Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Endometrial; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Ovarian Cancer; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Rat Breast; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Human Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Rat Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Paraformaldehyde-fixed, paraffin embedded Mouse Uterus; Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Antibody incubation with Phospho-Estrogen Receptor alpha (Ser118) Monoclonal Antibody, Unconjugated(bsm-52154R) at 1:200 overnight at 4°C, followed by conjugation to the bs-0295G-HRP and DAB (C-0010) staining.
Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Phospho-Estrogen Receptor alpha(S118)) monoclonal Antibody, Unconjugated (bsm-52154R) 1:50, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control:MCF7.
Primary Antibody (green line): Rabbit Anti-Phospho-Estrogen Receptor alpha (Ser118) antibody (bsm-52154R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1μg /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.