Sample:
HepG2 Cell Lysate at 40 ug
Eye (Mouse) Lysate at 40 ug
Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti- N-cadherin (bs-1172R)at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 100kD
Observed band size: 110kD

Sample:
Colon carcinoma(Human) lysate at 30ug;
Brain(Rat) lysate at 30ug;
Primary: Anti-CDH2/N-cadherin (bs-1172R) at 1:200 dilution;
Secondary: HRP conjugated Goat Anti-Rabbit IgG(bs-0295G-HRP) at 1: 3000 dilution;
Predicted band size : 100kD
Observed band size : 100kD

Sample:
Breast ca (Mouse) Lysate at 40 ug
Primary: Anti-N-cadherin (bs-1172R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 100 kD
Observed band size: 105 kD

Tissue/cell: human lung 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-N-cadherin Polyclonal Antibody, Unconjugated(bs-1172R) 1:400, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: rat brain 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-N-cadherin Polyclonal Antibody, Unconjugated(bs-1172R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: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 (N-cadherin) polyclonal Antibody, Unconjugated (bs-1172R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Tissue/cell: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 (N-cadherin) polyclonal Antibody, Unconjugated (bs-1172R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Tissue/cell: rat brain 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-N-cadherin Polyclonal Antibody, Unconjugated(bs-1172R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, PE conjugated(bs-0295G-PE)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: Hepg2(blue).
Primary Antibody:Rabbit Anti-E cadherin antibody (bs-1172R,Green); Dilution: 3μg 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.
Protocol
The cells were fixed with 2% paraformaldehyde for 10 min at 37℃. Primary antibody (bs-1172R, 3μg /1x10^6 cells) were incubated for 30 min at room temperature, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (1 hour) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/FITC antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 40 min at room temperature. Acquisition of 20,000 events was performed.