Sample:
Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon (SL0237R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 27 kD
Observed band size: 27 kD
Sample:Brain Cell Lysate at 40 ug
Primary: Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon (SL0237R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 27 kD
Observed band size: 27 kD
Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 ug
Lane 2: Uterus (Mouse) Lysate at 40 ug
Lane 3: Testis (Mouse) Lysate at 40 ug
Lane 4: Liver (Mouse) Lysate at 40 ug
Lane 5: Lung (Mouse) Lysate at 40 ug
Lane 6: Cerebrum (Rat) Lysate at 40 ug
Lane 7: Uterus (Rat) Lysate at 40 ug
Lane 8: Testis (Rat) Lysate at 40 ug
Lane 9: Liver (Rat) Lysate at 40 ug
Lane 10: Lung (Rat) Lysate at 40 ug
Lane 11: NIH/3T3 (Mouse) Cell Lysate at 30 ug
Lane 12: Siha (Human) Cell Lysate at 30 ug
Lane 13: U251 (Human) Cell Lysate at 30 ug
Primary: Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon (SL0237R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 28 kD
Observed band size: 28 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 (14-3-3 Alpha + Beta + Gamma + Delta + Epsilon) Polyclonal Antibody, Unconjugated (SL1024R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse 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 (14-3-3 Alpha + Beta + Gamma + Delta + Epsilon) Polyclonal Antibody, Unconjugated (SL1024R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Mouse 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 (14-3-3 Alpha + Beta + Gamma + Delta + Epsilon) Polyclonal Antibody, Unconjugated (SL0237R) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Tissue/cell: human rectal carcinoma;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-14-3-3 Polyclonal Antibody, Unconjugated(SL0237R) 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 (blue line): A549 (blue).
Primary Antibody (green line): Rabbit Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon antibody (SL0237R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 70% ethanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control:A431.
Primary Antibody (green line): Rabbit Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon antibody (SL0237R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
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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