Alexa fluor 546 goat anti mouse secondary

To evaluate YAP nuclear localization, MSCs at each time point were fixed in 4% paraformaldehyde for 30 minutes at 37°C and then permeabilized with 0.05% Triton X-100 in PBS supplemented with 320mM sucrose. Samples were then incubated with anti-YAP antibody in 1% BSA in PBS (1:200, sc-101199, Santa Cruz Bio, Dallas, Texas) overnight at 4°C, followed by incubation with Alexa-Fluor 546 goat anti-mouse secondary (1:200, A-11030, Molecular Probes, Grand Island, NY) at RT for 1 hour. To visualize cells, actin was stained with Alexa-Fluor 488 (1:1000, A12379, Molecular Probes, Grand Island, NY) for 30 minutes and 4′, 6-diamidino-2-phenylindole (DAPI, ProLong ® Gold antifade reagent with DAPI, P36935, Molecular Probes, Grand Island, NY) to stain nuclei. Using a fluorescence microscope with a 100× objective (Zeiss Axioplan-2 fluorescent microscope, Jena, Germany), images were taken and the average YAP staining intensity and localization were quantified using ImageJ (with nuclear staining intensity normalized to cytoplasmic staining) as in ^{22 (link),28 (link)}. To investigate the effect of acto-myosin contractility on YAP nuclear localization, MSCs cultured on TCP through P2 were treated with Y27632 (Y27, 10 μM, for 1 h, EMD Millipore, Bedford, MA) for 1 hour prior to fixation and imaging.