Mouse anti vinculin clone hvin 1

Human monocytes were isolated from blood of healthy donors as described previously (Van Goethem et al., 2010 (link)). Cells were re-suspended in cold PBS supplemented with 2 mM EDTA, 0.5% heat-inactivated fetal calf serum (FCS) at pH 7.4 and magnetically sorted with magnetic microbeads coated with antibodies directed against CD14 (Miltenyi Biotec). Monocytes were then seeded on glass coverslips at 1.5x10⁶ cells/well in six-well plates in RPMI 1640 (Invitrogen) without FCS. After 2 h at 37°C in humidified 5% CO₂ atmosphere, the medium was replaced by RPMI containing 10% FCS and 20 ng/mL of Macrophage Colony-Stimulating Factor (M-CSF) (Peprotech). For experiments, cells were used after seven days of differentiation.
Macrophages plated on glass coverslips (0.17 mm ±0.005mm, Marienfeld) were unroofed and fixed as previously described (Bouissou et al., 2017 (link)) and labelled with Alexa Fluor 488-phalloidin (Molecular Probes, 1/500) and Alexa Fluor 647-coupled phalloidin (Molecular Probes, A22287, 1/100) for RIM and dSTORM, respectively. Vinculin was stained using mouse anti-vinculin (clone hvin-1, Sigma-Aldrich, 1/500) and a secondary F(ab')₂ coupled to Alexa 555 (Cell Signaling technology).