Plan apochromat 40x 1.2 na water objective lenses

Cells were seeded on top of a collagen I matrix and immunostained as described^{12 (link)}. Cells were fixed with 4% formaldehyde, permeabilised with 0.3% Triton X-100, blocked with 5% bovine serum albumin (BSA), and stained with primary antibody pSer19-MLC2 (1:200, #3671, Cell Signalling), which was detected with secondary Alexa Fluor 488 or 647 antibodies (Life Technologies). F-actin was stained using Alexa Fluor 546-phalloidin (Life Technologies) and nuclei with Hoechst 33258 (Life Technologies). Antibodies were diluted in 5% BSA-PBS. Imaging was carried out on Zeiss LSM 510 Meta confocal microscope with Plan-Apochromat 40x/1.2 NA (water) objective lenses and Zeiss LSM 710 confocal microscope with Plan-Apochromat 40x/1.3 Oil or a Plan-Apochromat 63x/1.4 NA (oil) objective lenses (Carl Zeiss) and Zen software. Images were analysed using ImageJ. p-MLC2 fluorescence signal was quantified calculating the pixel intensity in single cells relative to the cell area.

Cells seeded on top of collagen matrices were fixed with 4% formaldehyde for 15 min at room temperature. Then, cells were permeabilised for 20 min using 0.3% Triton X-100 in 5% BSA-PBS, blocked in 5% BSA-PBS for 30 min and immunostained with primary antibody overnight at 4 °C. Next, samples were incubated with Alexa Fluor^TM 546-phalloidin (A22283, Thermo Fisher, 1:400) and Goat anti-rabbit Alexa Fluor^TM 488 (A-11008, Thermo Fisher, 1:1000) or Goat anti-mouse Alexa Fluor^TM 488 (A-11029, Thermo Fisher, 1:1000) secondary antibodies for 2 h at room temperature. Nuclei were stained with Hoechst prepared in PBS. Antibodies were diluted in 5% BSA-PBS.
Images were taken with a Zeiss LSM 510 Meta confocal microscope with Plan-Apochromat 40x/1.2 NA (water) objective lenses, Zeiss LSM 710 confocal microscope with Plan-Apochromat 40x/1.3 Oil DIC M27 and Zeiss LSM 880 confocal microscope with Airyscan superresolution mode and Plan-Apochromat 63x/1.4 Oil DIC M27 objective lenses (Carl Zeiss, Germany). Zen software was used to acquire images (Carl Zeiss, Germany). Images were analysed using ImageJ software (NIH). For pMLC2 quantification, fluorescence signal was quantified by calculating the area occupied by pMLC staining in single cells relative to the cell area. For pMLC2 distribution, line scan analysis was performed in Fiji 1.53t using the Plot profile plug in.