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Sample: Brain (Rat) Lysate at 40 ug Primary: Anti-Prostaglandin E Receptor EP1 (bs-6316R) at 1/300 dilution Secondary: HRP conjugated Goat-Anti-rabbit IgG (bs-0295G-HRP) at 1/5000 dilution Predicted band size: 42 kD Observed band size: 42 kD

Sample: Kidney (Mouse) Lysate at 40 ug Primary: Anti-Prostaglandin E Receptor EP1 (bs-6316R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 42 kD Observed band size: 45 kD

Tissue/cell: rat brain tissue;4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-PTGER1 Polyclonal Antibody, Unconjugated(bs-6316R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(bs-0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,C-0033) was used to stain the cell nuclei

Blank control (Black line): Mouse blood (Black). Primary Antibody (green line): Rabbit Anti-Prostaglandin E Receptor EP1 antibody (bs-6316R) Dilution: 3μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647 Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then 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.