Sample:
A431(Human) Cell Lysate at 30 ug
Primary: Anti-Phospho-RPS6 (Ser240 + Ser244) (bs-3389R) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 29 kD
Observed band size: 29 kD

Sample:
Jurkat(Human) Cell Lysate at 30 ug
Primary: Anti-Phospho-RPS6 (Ser240 + Ser244) (bs-3389R) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 29 kD
Observed band size: 29 kD

Paraformaldehyde-fixed, paraffin embedded (mouse cerebellum tissue); 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-RPS6(Ser240+Ser244)) Polyclonal Antibody, Unconjugated (bs-3389R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Tissue/cell: human lung carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Phospho-RPS6 (Ser240+Ser244) Polyclonal Antibody, Unconjugated(bs-3389R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Phospho-RPS6 (Ser240+Ser244) Polyclonal Antibody, Unconjugated(bs-3389R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control（black line）:HepG2.
Primary Antibody (green line): Rabbit Anti-Phospho-RPS6 (Ser240 + Ser244) antibody (bs-3389R)
Dilution:1ug/Test;
Secondary Antibody（white blue line）: Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/Test.
Isotype control（orange line）: Normal Rabbit IgG
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.