Sample: Lane 1: MCF-7 (Human) Cell Lysate at 30 ug Lane 2: MOLT-4 (Human) Cell Lysate at 30 ug Primary: Anti-Insulin Receptor alpha (SL0047R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 120 kD Observed band size: 120 kD
Sample:
Lane 1: MCF-7 (Human) Cell Lysate at 30 ug
Lane 2: Jurkat (Human) Cell Lysate at 30 ug
Lane 3: SW96 (Human) Cell Lysate at 30 ug
Lane 4: LOVO (Human) Cell Lysate at 30 ug
Primary: Anti-Insulin Receptor alpha (SL0047R) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 120 kD
Observed band size: 120 kD
Paraformaldehyde-fixed, paraffin embedded (human colon carcinoma); 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 (Insulin Receptor alpha) Polyclonal Antibody, Unconjugated (SL0047R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Tissue/cell: rat tongue 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,SLC0005) at 37℃ for 20 min;
Incubation: Anti- Insulin Receptor alpha Polyclonal Antibody, Unconjugated(SL0047R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control (blue line): HL60(blue).
Primary Antibody (green line): Rabbit Anti-Insulin Receptor alpha antibody (SL0047R)
Dilution: 0.2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 70% ethanol Overnight at 4℃. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 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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