Sample:
Brain(Rat)lysate at 30ug;
Heart(Rat)lysates, 30ug
Primary: Anti-BDNF (bs-0248R) at 1:200
Secondary: HRP conjugated Goat-Anti-Rabbit IgG(bse-0295G) at 1: 3000
Predicted band size : 13kD,25kD
Observed band size : 25kD

Tissue/cell: rabbit 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-BDNF Polyclonal Antibody, Unconjugated(bs-0248R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) 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; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min;
Incubation: Anti-BDNF Polyclonal Antibody, Unconjugated(bs-0248R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control: HepG2(blue).
Primary Antibody:Rabbit Anti-BDNF antibody (bs-0248R,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 (bs-0248R, 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.