Sample:
Lane 1: Lung (Rat) Lysate at 40 ug
Lane 2: Spleen (Rat) Lysate at 40 ug
Lane 3: MCF-7 (Human) Cell Lysate at 30 ug
Lane 4: Hela (Human) Cell Lysate at 30 ug
Primary: Anti-SCF (SL0545R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 45/31/19 kD
Observed band size: 45/35 kD
Paraformaldehyde-fixed, paraffin embedded (Rat brain); 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 (SCF) Polyclonal Antibody, Unconjugated (SL0545R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.
Tissue/cell: rat 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,SLC0005) at 37℃ for 20 min;
Incubation: Anti-SCF Polyclonal Antibody, Unconjugated(SL0545R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Tissue/cell: rat spleen 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-SCF Polyclonal Antibody, Unconjugated(SL0545R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Hela cell; 4% Paraformaldehyde-fixed; Ice-cold methanol at -20℃ for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (SCF) polyclonal Antibody, Unconjugated (SL0545R) 1:100, 90 minutes at 37°C; followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Tissue/cell: rat spleen 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,SLC0005) at 37℃ for 20 min;
Incubation: Anti-SCF Polyclonal Antibody, Unconjugated(SL0999R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, PE conjugated(SL0295G-PE)used at 1:200 dilution for 40 minutes at 37°C. DAPI(5ug/ml,blue,SLC0033) was used to stain the cell nuclei
Blank control (Black line): Mouse spleen(Black).
Primary Antibody (green line): Rabbit Anti-SCF antibody (SL0545R)
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 permeabilized with 90% ice-cold methanol 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 10,000 events was performed.
Blank control (Black line): U87MG (Black).
Primary Antibody (green line): Rabbit Anti-SCF antibody (SL0545R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE
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.
|