Anti fibronectin rabbit polyclonal

After 4 hours of culture, the culture medium was removed and unattached cells were washed away from the surface with PBS. Cells were fixed with 3 % paraformaldehyde in PBS for 30 min at 4 ⁰C, and washed three times with PBS. After permeabilizing with 0.1% Triton-X in PBS for 5 min at RT and blocking with DPBS/BSA 1 % at RT for 30 min, samples were incubated with a primary antibody for 1 hour at RT; the antibody used to analyze vinculin expression was antivinculin (mouse) (hVIN-1, Sigma-Aldrich) 1:400 in DPBS/BSA 1% and to analyze fibronectin expression was anti-fibronectin (rabbit) (polyclonal, Sigma-Aldrich) 1:400 in DPBS/BSA 1%.
Samples were washed twice with DPBS/Tween 20. A combination of secondary antibody (Cy3conjugated goat anti-mouse or antirabbit, respectively, Jackson ImmunoResearch) and phalloidin
(1:100) (BODIPY FL, Life Technology) was added and incubated by 1 hour at RT. Finally, after washing the samples with DPBS/Tween 20 three times, a mounting with Vectashield solution containing DAPI (Vector Laboratories) was performed.