Quantifying Filopodia-Associated Vinculin in CAD Cells

As described before^{20 (link)}, confluent CAD cells were mechanically detached and counted, and 300,000 cells were plated for 6 h per well in a 6 well plate. Cells were transfected as described above, using the abovementioned plasmids. At 24 h post-transfection, cells were detached and counted, and 140,000 cells were plated for 16 h on ibidi μ-dishes (ibidi GmbH). Cells were then fixed with 4% PFA for 20 min at RT and washed three times with PBS. Cells were then incubated for permeabilization and blocking with 2% BSA including 0.0075% saponin at RT for 1 h. Primary monoclonal antibody of vinculin (Sigma, 1:500) was prepared in PBS having 2% BSA and 0.01% saponin and incubated at RT for 1 h. After several washes with PBS cells were incubated with secondary antibody goat anti-mouse Alexa Fluor 546 (Invitrogen, Thermo Fisher Scientific) in the same solution at RT for 1 h. Cells were then stained with HCS Cell Mask Blue Stain (Invitrogen, Thermo Fisher Scientific, 1:5000) in PBS for 20 min, washed several times and mounted. Quantification was performed as described before^{20 (link),30 (link),32 (link)} by (1) creating the ROI restricted to the outer region of the cells that covers only attached filopodia; (2) automatized counting of the vinculin positive filopodia using spot detector tool (ICY software).