Paraformaldehyde-fixed, paraffin embedded (Rat skeletal muscle); 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 (IRS-2) Polyclonal Antibody, Unconjugated (bs-20748R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat kidney tissue); 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 (IRS-2) Polyclonal Antibody, Unconjugated (bs-20748R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Rat brain); 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 (IRS-2) Polyclonal Antibody, Unconjugated (bs-20748R) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

SH-SY5Y cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (IRS-2) polyclonal Antibody, Unconjugated (bs-20748R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control（black line）:SH-SY5Y.
Primary Antibody (green line): Rabbit Anti-IRS-2 antibody (bs-20748R)
Dilution:1ug/Test;
Secondary Antibody（white blue line）: Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/Test.
Isotype control（orange line）: Normal Rabbit IgG
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.

Blank control (Black line): Raji (Black).
Primary Antibody (red line): Rabbit Anti-IRS-2 antibody (bs-20748R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (green line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA （10min）and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature . 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.

Blank control (black line): HepG2(black) (The cells were fixed with 2% paraformaldehyde (10 min) , then permeabilized with PBST for 30 min on room temperature)
Primary Antibody (Red line): Rabbit Anti-IRS-2 antibody (bs-20748R) ; Dilution: 1μg /10^6 cells;
Isotype Control Antibody (green line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC;Dilution: 1μg /test.