Immunofluorescence for Fibronectin

1.5 x 10⁵ HDF were seeded on glass slides in 6-well plates and treated with TVE and the control for 72 hrs. After the treatments, the medium was removed and the cells were washed with PBS 1x, fixed in paraformaldehyde (PFA) 4% for 10ʹ, washed three times with PBS 1x, and permeabilized with 1% Triton X-100 in PBS for 30ʹ. The cells were then treated for 30ʹ with 0.5% Tween and 5% BSA in PBS and incubated overnight at 4°C with primary mouse monoclonal antibody, raised against human fibronectin (A-11) (Santa Cruz Biotechnology) diluted 1:200 in PBS 1x, containing 0.5% Tween and 1% BSA. After incubation, samples were washed 3 times with PBS + 0.5% Tween 20, incubated with anti-mouse secondary antibody, labelled with Alexa Fluo 488 (Thermo Scientific) and diluted 1:1000 in PBS 1x, containing 0.5% Tween 20 and 1% BSA in presence of DAPI 0.5ug/mL. One hour later, the slides were washed 3 times with PBS + 0.5% Tween 20 and the slides were mounted on coverslip using PBS with 50% of glycerol. The intensity of fluorescence was measured using ImageJ software and the data reported in the graph represented the ratio between the fluorescence intensity and the number of nuclei.