Tissue/cell: human brain glioma; 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-EX33 Polyclonal Antibody, Unconjugated(bs-15353R) 1:500, overnight at 4∑C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: human colon carcinoma; 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-GPCR EX33 Polyclonal Antibody, Unconjugated(bs-15353R) 1:500, overnight at 4∑C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

araformaldehyde-fixed, paraffin embedded (rabbit thymus); 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 (GPCR EX33) Polyclonal Antibody, Unconjugated (bs-15353R) at 1:500 overnight at 4∑C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Human Brain Glioma); 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 (GPCR EX33) Polyclonal Antibody, Unconjugated (bs-15353R) at 1:500 overnight at 4∑C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Tissue/cell: rabbit thymus 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-EX33 Polyclonal Antibody, Unconjugated(bs-15353R) 1:500, overnight at 4∑C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control:U937.
Primary Antibody (green line): Rabbit Anti-GPR84 antibody (bs-15353R)
Dilution: 1ug/Test;
Secondary Antibody (white blue line) : Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/Test.
Isotype control（orange line）：Normal Rabbit IgG
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.

Blank control(blue): Mouse thymus cells(fixed with 2% paraformaldehyde (10 min)).
Primary Antibody:Rabbit Anti- GPCR EX33 antibody(bs-15353R), 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.