MCF7 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 (MUC1) monoclonal Antibody, Unconjugated (SLM51126M) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Mouse IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control(black line):MCF-7.
Primary Antibody (green line): Mouse Anti-MUC1 antibody (SL52116M)
Dilution:1ug/Test;
Secondary Antibody(white blue line): Goat anti-Mouse IgG-AF488
Dilution: 0.5ug/Test.
Isotype control(orange line): Normal Mouse IgG
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃, 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.