Immunocytochemistry

Detailed protocols for fixation and staining of the cells have been described previously (Lewandowska et al., 2015). Briefly, medium was removed, and cells were washed with PBS. Cells were then fixed in 4% paraformaldehyde for 15 min, and washed with cold PBS. Cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min and then washed. Unspecific binding was blocked by incubating cells in 1% BSA in PBS for 30 min. Cells were then incubated with primary antibodies (desmin, ab32362, Abcam; sarcomeric α-actinin, ab9465, Abcam) overnight at 4°C, washed, and then incubated with secondary antibodies (goat anti-mouse IgG AlexaFluor 488, A-11001, ThermoFisher and donkey anti-rabbit IgG AlexaFluor 555, ab150074, Abcam) for 1 h. After washing again, the cells were incubated with DAPI for 1 min, rinsed with PBS, and left in PBS at 4°C. Imaging was done using a Nikon Eclipse LVDIA-N microscope.