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-DRD1 Polyclonal Antibody, Unconjugated(bs-1007R) 1:300, overnight at 4∑C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control: Hela(blue).
Primary Antibody:Rabbit Anti- Dopamine Receptor D1 antibody(bs-1007R), Dilution: 1ug in 100 ul 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) , then permeabilized with 90% ice-cold methanol for 30 min on ice. Antibody (bs-1007R, 1ug /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 10% 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 bs-1007R at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.