Paraformaldehyde-fixed, paraffin embedded (Human brain glioma); 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; Incubation: Anti-EGFR Antibody, conjugated (SLM33050M-FITC EGFR) 1:400, 1.5 hours at 37°C; DAPI (5ug/ml, blue, SLC0033) was used to stain the cell nuclei.
Blank control:Hela.
Primary Antibody (green line): Mouse Anti-EGFR antibody (SLM33050M-FITC)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): 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. Acquisition of 20,000 events was performed.
Blank control:A549.
Primary Antibody (green line): Mouse Anti-EGFR antibody (SLM33050M-FITC)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): 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. Acquisition of 20,000 events was performed.