Mouse anti β tubulin t4026

Fixed cells were permeabilized for 4 min in TBS (50 mM Tris-HCl, 0.15 M NaCl, pH 7.4) containing 0.2% Triton X-100. After rinsing with TBS, the cell samples were blocked for 1h at room temperature with 2% BSA (900.001, Aurion) in TBS. The samples were incubated for 1h at room temperature with the primary antibodies against BMP-2 (mouse anti-BMP-2, B9553Sigma, at 2.5 µg/mL), rab7 (rabbit anti-rab7, D95F2 Abcam, dilution 1:50), cav-1 (rabbit anti-cav-1, sc 894, Santa Cruz at 2 µg/mL), tubulin (mouse anti-β-tubulin, T4026, Sigma 1:200) and pSMAD1/5/8 (1:600, Cell Signaling). A 0.2% gelatin (G7765, Sigma) in TBS solution was used as incubation buffer. After extensive washing with TBS, the cells were further incubated with goat anti-mouse A647 (A21335, Invitrogen) or goat anti-rabbit A647 (A21244, Invitrogen) secondary antibodies diluted to 1:200 in 0.2% gelatin in TBS for 1h at room temperature. Actin was labeled with phalloidin-TRITC (1:800, Sigma) for 30 min. The cell nuclei were stained with 5 µg/mL of DAPI (Life Technologies) for 10 min. Nuclear p-SMAD1/5/8 intensity was measured over nucleus area, obtained from binarized nucleus images, using homemade Image J (National Institutes of Health) routines.