Sample:
Lane 1: Heart (Mouse) Tissue Lysate at 40 ug
Lane 2: Bone (Mouse) Tissue Lysate at 40 ug
Lane 3: Heart (Rat) Tissue Lysate at 40 ug
Primary:
Anti-Adenosine A3 Receptor (SL1225R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 35 kD
Observed band size: 36 kD
Tissue/cell: human gastric 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,SLC0005) at 37℃ for 20 min;
Incubation: Anti-ADORA3 Polyclonal Antibody, Unconjugated(SL1225R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Paraformaldehyde-fixed, paraffin embedded (rat 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 (Adenosine A3 Receptor) Polyclonal Antibody, Unconjugated (SL1225R) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Blank control (blue line): Raji (blue)(The cells were fixed with 70% ethanol overnight at -20℃). Primary Antibody (green line): Rabbit Anti-Adenosine A3 Receptor antibody(SL1225R); Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE;Dilution: 1μg /test.
Blank control (blue line): Raji (blue)(The cells were fixed with 70% ethanol overnight at -20℃). Primary Antibody (green line): Rabbit Anti-Adenosine A3 Receptor antibody(SL1225R); Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE;Dilution: 1μg /test.
Blank control:HL-60.
Primary Antibody (green line): Rabbit Anti-Adenosine A3 Receptor antibody (SL1225R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /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.
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