Blank control: Molt-4. Primary Antibody (green line): Rabbit Anti-phospho-eIF4E (Ser209) antibody (orb13390), Dilution: 0.2 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-PE, Dilution: 0.2 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 20% PBST for 20 min at -20C. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at 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 (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 37C for 30 min, Antibody incubation with (phospho-eIF4E (Ser209)) Polyclonal Antibody, Unconjugated (orb13390) at 1:400 overnight at 4C, 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 37C for 30 min, Antibody incubation with (phospho-eIF4E (Ser209)) Polyclonal Antibody, Unconjugated (orb13390) at 1:400 overnight at 4C, 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 37C for 30 min, Antibody incubation with (phospho-eIF4E (Ser209)) Polyclonal Antibody, Unconjugated (orb13390) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: NIH/3T3 (Mouse) Cell Lysate at 30 ug, Primary: Anti-phospho-eIF4E (Ser209) (orb13390) at 1/500 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 24 kD, Observed band size: 28 kD.
IHC-P analysis of Rat brain using phospho-eIF4E Ser209 Polyclonal Antibody at 1:400 dilution.
IHC-P analysis of Mouse brain using phospho-eIF4E Ser209 Polyclonal Antibody at 1:400 dilution.
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