Sample:
Liver(Mouse) Lysate at 40 ug
Primary: Anti-PODXL (SL1345R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 59 kD
Observed band size: 59 kD
Sample:Kidney(Mouse) lysate at 30 ug;
Primary: Anti-PODXL (SL1345R) at 1:300 dilution;
Secondary: HRP conjugated Goat-Anti-Rabbit IgG(bse-0295G-HRP) at 1: 5000 dilution;
ECL excitated the fluorescence;
Predicted band size : 59 kD
Observed band size :59 kD
Tissue/cell: human 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,SLC0005) at 37℃ for 20 min;
Incubation: Anti-PCX Polyclonal Antibody, Unconjugated(SL1345R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control: RSC96(blue). Primary Antibody:Rabbit Anti- PODXL antibody(SL1638R), Dilution: 0.2μg 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-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) . Antibody (SL1345R, 0.2μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody of SL1345R at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.
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