Sample:
Lane 1: Mouse Liver Lysates
Lane 2: Mouse Brain Lysates
Lane 3: Human HepG2 cell Lysates
Lane 4: Human K562 cell Lysates
Lane 5: Human A549 cell Lysates
Primary: Anti-Frizzled 5 (SL2930R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 62kDa
Observed band size: 62kDa
Sample:
Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti- Frizzled 5 (SL2930R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 62 kD
Observed band size: 62 kD
Images provided the Independent Validation Program (badge number 28751)Formalin-fixed and paraffin embedded mouse heart labeled with Rabbit Anti-Frizzled 5 Polyclonal Antibody (SL2930R) at 1:250 room temperature overnight followed by conjugation to secondary antibody.
Tissue/cell: mouse pancreas 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-Frizzled 5 Polyclonal Antibody, Unconjugated(SL2930R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Tissue/cell: mouse embryos 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-Frizzled 5 Polyclonal Antibody, Unconjugated(SL2930R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control:K562.
Primary Antibody (green line): Rabbit Anti-Frizzled 5 antibody (SL2930R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-PE
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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