Phospho-p38 (T180 + Y182) Rabbit Polyclonal Antibody

Sample:
Muscle (Mouse) Lysate at 40 ug
Muscle (Rat) Lysate at 40 ug
Primary: Anti-Phospho-P38 MAPK (Thr180 + Tyr182) (ER1903-01) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 kD

Sample:
Kidney (Mouse) Lysate at 40 ug
Primary: Anti-Phospho-P38 MAPK (Thr180 + Tyr182) (ER1903-01) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 42 kD
Observed band size: 42 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; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-P38 MAPK(Phospho-Thr180/Tyr182) Polyclonal Antibody, Unconjugated (ER1903-01) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: human placenta 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-P38 MAPK(Phospho-Thr180/Tyr182) Polyclonal Antibody, Unconjugated (ER1903-01) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Tissue/cell: mouse 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-P38 MAPK(Phospho-Thr180/Tyr182) Polyclonal Antibody, Unconjugated (ER1903-01) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Paraformaldehyde-fixed, paraffin embedded (rat brain tissue); 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 (P-P38 MAPK) Polyclonal Antibody, Unconjugated (ER1903-01) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Blank control: HepG2(blue). Primary Antibody:Rabbit Anti-Phospho-P38 MAPK (Thr180 + Tyr182)antibody (ER1903-01,Green); Dilution: 1μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions; Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.

Protocol
The cells were fixed with 2% paraformaldehyde for 10 min at 37℃. Primary antibody (ER1903-01, 1μg /1x10^6 cells) were incubated for 30 min at room temperature, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/FITC antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 40 min at room temperature. Acquisition of 20,000 events was performed.