following protocol used human PBMC that were isolated from whole human blood
(Ficoll method) and plated onto fibronectin-coated coverslips in 10% FCS/DMEM
supplemented with pen/strep and L-glutamine. Cells were allowed to adhere at 37˚C
for 1 hour before being fixed in 4% paraformaldehyde for 20 minutes at room
cells were permeabilized with 0.1% Triton X-100/PBS for 2 minutes before being
washed extensively with PBS.
were blocked with 1% BSA/PBS.
were stained with the primary antibody (diluted in 1%BSA/PBS) at room
temperature for 1 hour.
were stained with the secondary antibodies for cytoskeleton and nuclei
were mounted onto glass slides with medium and imaged.