Immunofluorescence Analysis of Myosin and Ki67

Cells were fixed with 4% PFA in PBS for 10 min at 4°C and processed for immunofluorescence analysis as previously described (Testa et al., 2017 (link)). Briefly, cells were washed with PBS and blocked with 10% goat serum in PBS for 1 h at RT. Subsequently, cells were incubated with the primary antibody anti-myosin heavy chain (MF20, mouse monoclonal, DHSB, diluted 1:2) or anti-ki67 (rabbit polyclonal, Novus Biologicals #NB110-89717, diluted 1:200) for 1 h, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L; Thermo Fisher Scientific #A21422, diluted 1:400) and 488-conjugated goat anti-rabbit IgG (H + L; Thermo Fisher Scientific #A11008, diluted 1:400) for 1 h. Finally, nuclei were stained with 300 nM DAPI (Thermo Fisher Scientific) for 10 min. Specimens were viewed using a Nikon TE 2000 epifluorescence microscope equipped with a Photometrics Cool SNAP MYO CCD camera.