Tcs sp5 fluorescent microsystems

Images were captured by Leica TCS SP5 fluorescent microsystems using appropriate filters. All images were taken with the same exposure time and the same threshold to allow subsequent quantitative analysis in the respective channel. At least 150 nuclei per sample were imaged and the images were processed with ImageJ. Statistical analyses were then done by Blobfinder software (available for download from the Centre of Image Analysis at Uppsala University, http://www.cb.uu.se/~amin/BlobFinder) to determine the total fluorescence intensity per cell (Lipovsky et al. 2015 (link)). Experiments were repeated three times and averaged to obtain mean ± SEM. There is a low, variable background in these experiments, which is typically <10% of the signal deemed positive.

HeLa cells grown on glass coverslips were treated with 5 µg/mL ActD or vehicle control for 1 h, fixed with 4% paraformaldehyde in PBS, and permeabilized with 1% saponin in PBS. After incubation in blocking buffer (DMEM containing 10% FBS) for 1 h at room temperature, samples were stained with a 1:1000 dilution of anti-HuR mouse antibody followed by incubation with 1:200 dilution of the AlexaFluor 488 goat anti-mouse antibody (Molecular Probes). Both antibodies were diluted in blocking buffer. The nuclei were stained with DAPI (blue). The images were taken by Leica TCS SP5 fluorescent microsystems and processed with ImageJ.