Tissue/cell: HeLa cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (Kir6.2) Polyclonal Antibody, Unconjugated (SL2436R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody (SL0295G-FITC) at 37°C for 90 minutes, DAPI (5ug/ml, blue, SLC0033) was used to stain the cell nuclei.
Blank control: A549.
Primary Antibody (green line): Rabbit Anti-Kir6.2 antibody (SL2436)
Dilution: 3μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-PE
Dilution: 1μg /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.