Lsm 880 spectral

We seeded U2OS cells on glass coverslips and performed fixation by incubating cells at room temperature for 20 min in 4% paraformaldehyde. Then, cells were permeabilised for 20 min with 0.05% saponin in phosphate-buffered saline containing 0.5% bovine serum albumin. The primary antibody, anti-phospho-p38 (Cell signalling #9211) (1:200), was incubated at 37 °C for 1 h. After washing, Alexa-Fluor 488 secondary antibody (Invitrogen) (1:500) was incubated at 37 °C for 45 min. Actin filaments were stained by incubation with phalloidin-Alexa 647 (BioProbes) (100 ng/mL) for 20 min at room temperature. Nuclei were stained with DAPI (Sigma-Aldrich) (1 μg/mL). All images were acquired using a confocal laser scanning microscope (LSM 880 spectral, Carl Zeiss Microscopy GmbH, Jena, Germany).

U2OS cells grown on glass coverslips were fixed at room temperature for 20 min with 4% paraformaldehyde. Cells were permeabilized for 20 minutes with 0.05% saponin in PBS containing 0.5% bovine serum albumin. The primary antibody, anti-p-p38 (1:200), was incubated at 37 °C for 1 hour. After washing, Alexa-Fluor 488 secondary antibody (1:500) was incubated at 37 °C for 45 minutes. Incubation with phalloidin-Alexa 647 (100 ng/mL) for 20 minutes at room temperature was used to detect F-actin. Nuclei were stained with DAPI (1 μg/mL). Images were acquired using a confocal laser scanning microscope (LSM 880 spectral, Carl Zeiss Microscopy GmbH, Jena, Germany).