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Tissue/cell: human kidney 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) at 37℃ for 20 min; Incubation: Anti-phospho-CREB-1(Ser133) Polyclonal Antibody, Unconjugated 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAB staining
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) at 37℃ for 20 min; Incubation: Anti-phospho-CREB-1(Ser133) Polyclonal Antibody, Unconjugated 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugatedCy3)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue) was used to stain the cell nuclei
Blank control: RSC96(blue), the cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice. Isotype Control Antibody: Rabbit IgG(orange) ; Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA ; Primary Antibody Dilution: 1ug in 100 uL1X PBS containing 0.5% BSA(green).
Sample: Lane 1: Cerebrum (Mouse) Lysate at 40 ug Lane 2: Cerebellum (Mouse) Lysate at 40 ug Lane 3: Kidney (Mouse) Lysate at 40 ug Lane 4: Huvec (Human) Cell Lysate at 30 ug Primary: Anti-phospho-CREB-1 (Ser133) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 43 kD Observed band size: 45 kD
