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Sample:
MDCK(Dog) Cell Lysate at 40 ug
Primary: Anti-Nephrin (bs-10233R) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 138 kD
Observed band size: 138 kD

Sample:
Liver (Mouse) Lysate at 40 ug
Primary: Anti-Nephrin (bs-10233R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 138 kD
Observed band size: 138 kD

Tissue/cell: mouse kidney 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-Nephrin Polyclonal Antibody, Unconjugated(bs-10233R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: rat kidney 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-Nephrin Polyclonal Antibody, Unconjugated(bs-10233R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control: RSC96(blue), the cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice.
Isotype Control Antibody: Rabbit IgG(orange) ; Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA ; Primary Antibody Dilution: 1μg in 100 μL1X PBS containing 0.5% BSA(green).

the cells(293T) were fixed with 2% paraformaldehyde (10 min).
Isotype Control Antibody: Rabbit IgG(orange) ; Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA ; Primary Antibody Dilution: 1μg in 100 μL1X PBS containing 0.5% BSA(green).

Blank control:293T.
Primary Antibody (green line): Rabbit Anti-Nephrin antibody (bs-10233R)
Dilution: 1ug/Test;
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 0.5ug/Test.
Protocol
The cells wereincubated 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.