Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 ug
Lane 2: Cerebrum (Rat) Lysate at 40 ug
Primary: Anti-5HT2A Receptor (SL1056R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 53 kD
Observed band size: 50 kD
Sample:
U251(Human) Cell Lysate at 30 ug
BV2(Mouse) Cell Lysate at 30 ug
Primary: Anti-5HT2A Receptor (SL1056R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kD
Observed band size: 52 kD
Tissue/cell: rat lung 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-5HT2A Receptor Polyclonal Antibody, Unconjugated(SL1056R) 1:200, overnight at 4°C; The secondary antibody was Goat Anti-Rabbit IgG, Cy3 conjugated(SL0295G-Cy3)used at 1:200 dilution for 40 minutes at 37°C.
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 (5HT2A Receptor) Polyclonal Antibody, Unconjugated (SL1056R) at 1:400 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (SL0295G- cy3) for 90 minutes, and DAPI for nuclei staining.
Blank control: U-2OS.
Primary Antibody (green line): Rabbit Anti-5HT2A Receptor antibody (SL1056R)
Dilution: 3μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 3μg /test.
Protocol
The cells were incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at 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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