Bx51 upright fluorescent microscope

A549 cells were plated on glass cover-slips in 6 well plates and transfected with 100 nM siRNA, serum starved over-night, treated with 10 ng/mL TGFβ for 1 hour and processed for immunofluorescence. For the inhibitor studies, cells were plated on glass-cover slips, serum starved for 24 hours and treated with 10 ng/mL TGFβ in presence of 10 µM inhibitors (SB431542 or 2OH-BNPP1) for 1 hour. Cells on cover-slips were washed with PBS and fixed in 10% (v/v) buffered-formalin (5 min at room temperature, RT) followed by methanol fixation at −20°C for 20 min. Cover-slips were rehydrated by incubating 3 times in PBS for 5 min and permeabilized in 0.2% (v/v) Triton X100- PBS containing 1% (w/v) BSA on ice for 5 min. Cover slips were blocked by 5% (w/v) BSA, 5% (v/v) goat serum, 10% (v/v) donkey serum in PBS for 1 hour. Anti-SMAD2 antibody at 1:200 dilution was added in PBS containing 0.5% (w/v) BSA and incubated for 1 hour at 37°C in humidified chamber. Cover glasses were washed 3 times in PBS containing 0.025% (v/v) Triton X-100 and incubated with anti-Rabbit-488 secondary antibody for 1 hour. After washing, cover slips were mounted in ProLongGold (Invitrogen) containing DAPI, dried overnight at room temperature and stored at −20°C until microscopy. Micrographs were taken using Olympus BX-51 upright fluorescent microscope fitted with an Olympus DP-70 high resolution digital color camera.