Tcs sted cw sp8 super resolution microscope

Cells were grown on 24 well plates with round glass coverslips (Thermo Scientific) followed by fixation in ice-cold methanol. Afterwards, samples were blocked in PBS/3% w/v BSA for 30 min and stained with anti-smooth muscle myosin heavy chain 11 antibody for 1 h at room temperature. Cells were washed with PBS and incubated with Alexa Fluor^®488 goat anti-rabbit IgG (H + L) secondary antibody for 45 min at room temperature in darkness and DNA was counterstained with 4′,6-diamino-2-fenilindol (DAPI). After three washes coverslips were mounted in Fluoromount™ Aqueous Mounting Medium (Sigma-Aldrich, St Louis, MO, USA). Image acquisition was performed with Leica TCS STED CW SP8 super-resolution microscope with a 40x or 10x lens and recording optical sections every 0.3 µm. Image analysis was done using ImageJ software (National Institute of Health, USA).

Cells were grown on round 14 mm glass coverslips (Thermo Scientific, Waltham, MA, USA), fixed in ice-cold methanol for 10 min at room temperature and then blocked in PBS/3% w/v BSA (A6003, Sigma-Aldrich, St. Louis, MO, USA) for 30 min and stained with primary antibody anti-smooth muscle Myosin heavy chain 11 (ab82541, Abcam, Cambridge, UK) for 1 h at room temperature. Cells were washed in PBS pH 7.45 and incubated with Alexa Fluor^® 488 goat anti-rabbit IgG (H + L) (A-11008, Invitrogen, Thermofisher, Carlsbad, CA, USA) secondary antibody for 45 min at room temperature in darkness and DNA was counterstained with 4′,6-diamino-2-fenilindol (DAPI). After three washes, coverslips were mounted in Fluoromount™ Aqueous Mounting Medium (F4680, Sigma-Aldrich, St. Louis, MO, USA). Image acquisition was performed with Leica TCS STED CW SP8 Super-Resolution Microscope with a 40× lens and recording optical sections every 0.3 µm. Image analysis was done analyzing MYH11 staining fluorescence of each cell per cell area in MIT and PLQ VSMCs with ImageJ software (National Institute of Health, Bethesda, MD, USA).