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PubMed:ISSN:1936-2625/IJCEP0023819

Tissue/cell: human kidney 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-IRS-1(Ser307) Polyclonal Antibody, Unconjugated(bs-2736R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control (blue line): Hela (fixed with 70% ethanol (Overnight at 4℃) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature).
Primary Antibody (green line): Rabbit Anti-phospho-IRS1 (Ser307) antibody (bs-2736R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
Dilution: 1μg /test.

Blank control: HepG2.
Primary Antibody (green line): Rabbit Anti-phospho-IRS1 (Ser307) antibody (bs-2736R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
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.