Alexa 568 conjugated donkey anti mouse igg

HLE-B3 cells were grown on coverslips in 60-mm culture dishes and transfected in parallel with FLAG constructs corresponding to wild-type and six mutant FOXE3 alleles, with equal amounts of plasmid DNA per dish. After 48 h, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized with 0.25% Triton X-100 PBS for 5 min. Coverslips were blocked in 10% normal goat serum in PBS at 37°C for 30 min, incubated overnight with anti-FLAG M2 antibody (1:500, Sigma-Aldrich), washed with PBS three times, incubated for 1 h with Alexa 568-conjugated donkey antimouse IgG (1:1000, Thermo Fisher), washed with PBS, mounted on glass slides with PermaFluor aqueous media (Thermo Fisher) containing 1 μg/ml 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) and imaged with a fluorescence microscope. The numbers of cells with cytoplasmic and nuclear-restricted FLAG staining were counted in 10 randomly selected fields and averaged for three independent transfections per construct, with untransfected HLE-B3 cells as a negative control. The percentage of FLAG+ cells with cytoplasmic staining was plotted for each construct using GraphPad Prism 9. Statistical significance was assessed using an unpaired t-test. Results are given as mean ± SEM.