A549 cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37C for 20 min, Antibody incubation with (phospho-IRS1 (Ser616)) polyclonal Antibody, Unconjugated (orb184392) 1:100, 90 minutes at 37C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.
Blank control (blue line): Hela (fixed with 70% ethanol (Overnight at 4C) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature). Primary Antibody (green line): Rabbit Anti-phospho-IRS1 (Ser616) antibody (orb184392), dilution: 0.2 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE, dilution: 1 µg/Test.
Blank control: HepG2. Primary Antibody (green line): Rabbit Anti-phospho-IRS1 (Ser616) antibody (orb184392), 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 -20C. 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 (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-IRS1 (Ser616)) Polyclonal Antibody, Unconjugated (orb184392) at 1:400 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat uterus), 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-IRS1 (Ser616)) Polyclonal Antibody, Unconjugated (orb184392) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: Lane 1: Mouse Cerebrum tissue lysates, Lane 2: Mouse Cerebellum tissue lysates, Primary: Anti-phospho-IRS1 (Ser616) (orb184392) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 131 kDa, Observed band size: 185 kDa.
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