Paraformaldehyde-fixed, paraffin embedded (Mouse 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 (Phospho-TrkA(Tyr496) +TrkB(Tyr516)) Polyclonal Antibody, Unconjugated (SL3456R) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Blank control: K562.
Primary Antibody (green line): Rabbit Anti-Phospho-TrkA (Tyr496)+TrkB(Tyr516)antibody (SL3456R)
Dilution: 1ug/Test;
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 0.5ug/Test.
Protocol
The cells were 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.