Alexafluor 488 goat anti mouse igg1

Cell adhesion onto the alloy surface was analysed using an antibody against vinculin to determine the presence of focal contacts. At the same time, phalloidin was used to visualize actin filaments and their distribution, as reported previously [8 (link)]. The same cell culture protocol described for viability studies was employed, but after 24 h of culture the cells were fixed in 4% PFA in PBS for 30 min at RT, permeabilised with 0.1% Triton X-100 (Sigma) in PBS for 15 min and blocked for 25 min with 1% bovine serum albumin (BSA; Sigma) in PBS at RT. Samples were then incubated with 2 μg/ml mouse anti-vinculin primary monoclonal-antibody (Chemicon, MAB3574) for 60 min at RT and washed with 1% BSA-PBS. Then, samples were incubated with a mixture of 1.4 U/ml Alexa fluor 594-conjugated phalloidin (Invitrogen), 6 μg/ml Alexa fluor 488 goat anti-mouse IgG1 and Hoechst 33258 (both from Sigma) for 60 min at RT. Finally, samples were washed in 1% BSA-PBS, air dried and mounted on specific bottom glass dishes (MatTek) using ProLong mounting solution (Life Technologies). Control analyses were performed in absence of the alloy. Sample evaluation was done with a confocal laser scanning microscope (CLSM, Olympus). One disk was analysed for each surface modification and alloy composition.