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,SLC0005) at 37℃ for 20 min;
Incubation: Anti-phospho-IGF1R(Tyr196) Polyclonal Antibody, Unconjugated(SL5447R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Paraformaldehyde-fixed, paraffin embedded (human stomach); 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 (RELM beta) Polyclonal Antibody, Unconjugated (SL5774R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
The blue histogram is unstained cells(Hepg2 cells).
The green histogram is cells stained with Rabbit Anti-phospho-IGF1R (Tyr196) antibody (SL5447R) plus secondary antibody(SL0295G-FITC).Concentration 1:20
Blank control(blue): Mouse Kidney (fixed with 2% paraformaldehyde for 10 min at 37℃).
Primary Antibody:Rabbit Anti-phospho-IGF1R (Tyr196) antibody (SL5447R,Green); Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA;
Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions;
Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Blank control: MCF7.
Primary Antibody (green line): Rabbit Anti-phospho-IGF1R (Tyr196) antibody (SL5447R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were 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.
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