Paraformaldehyde-fixed, paraffin embedded (rat ovary); 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 (p-SMAD5) Polyclonal Antibody, Unconjugated (SL19918R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Blank control（black line）:HepG2.
Primary Antibody (green line): Rabbit Anti-phospho-SMAD5 (Ser463 + Ser465) antibody (SL19918R)
Dilution:1ug/Test;
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/Test.
Negative control（white blue line）: PBS
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.
