Sample:
Lane 1: Mouse Placenta tissue lysates
Lane 2: Mouse Breast tissue lysates
Lane 3: Mouse Testis tissue lysates
Lane 4: Rat Placenta tissue lysates
Lane 5: Rat Breast tissue lysates
Lane 6: Rat Testis tissue lysates
Lane 7: Human MCF-7 cell lysates
Lane 8: Human HepG2 cell lysates
Lane 9: Human A431 cell lysates
Primary: Anti- Amphiregulin (bs-3847R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 16 kDa
Observed band size: 47 kDa

Sample:
HepG2(Human) Cell Lysate at 30 ug
Primary: Anti-Amphiregulin (bs-3847R) at 1/2000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 26 kD
Observed band size: 41 kD

Sample:
A549(Human) Cell Lysate at 30 ug
Primary: Anti-Amphiregulin (bs-3847R) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 26 kD
Observed band size: 41 kD

Paraformaldehyde-fixed, paraffin embedded (human gastric 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 (Amphiregulin) Polyclonal Antibody, Unconjugated (bs-3847R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human tonsil); 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 (Amphiregulin) Polyclonal Antibody, Unconjugated (bs-3847R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human liver 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 (Amphiregulin) Polyclonal Antibody, Unconjugated (bs-3847R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions 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 (Amphiregulin) Polyclonal Antibody, Unconjugated (bs-3847R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.

Tissue/cell: mouse embryo tissue;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Amphiregulin Polyclonal Antibody, Unconjugated(bs-3847R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Tissue/cell: mouse fetal liver;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Amphiregulin Polyclonal Antibody, Unconjugated(bs-3847R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Tissue/cell: mouse fetal intestine;4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-Amphiregulin Polyclonal Antibody, Unconjugated(bs-3847R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Blank control:A549.
Primary Antibody (green line): Rabbit Anti-Amphiregulin antibody (bs-3847R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-PE
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 20% PBST for 20 min at-20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at 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.