Sample:
Spleen (Mouse) Lysate at 40 ug
LOVO Cell (Human) Lysate at 40 ug
HL60 Cell (Human) Lysate at 40 ug
U937 Cell (Human) Lysate at 40 ug
MCF-7 Cell (Human) Lysate at 40 ug
Hela Cell (Human) Lysate at 40 ug
Primary: Anti- Phospho-Bcl2 (Thr69) (SL12578R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 26 kD
Observed band size: 26 kD
Paraformaldehyde-fixed, paraffin embedded (rat stomach 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-Bcl2 (Thr69)) Polyclonal Antibody, Unconjugated (SL12578R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Tissue/cell: rat lung 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,SLC0005) at 37℃ for 20 min;
Incubation: Anti-Phospho-Bcl2 (Thr69) Polyclonal Antibody, Unconjugated(SL12578R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control(blue):K562 (fixed with 2% paraformaldehyde for 10 min at 37℃). Primary Antibody:Rabbit Anti-Phospho-Bcl2 (Thr69) antibody (SL12578R,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.
Blank control:HL-60.
Primary Antibody (green line): Rabbit Anti-Phospho-Bcl2 (Thr69) antibody (SL12578R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF488
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 0.1%PBST for 20 min at room temperature. The cells were then 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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