Nsc 2025

HDF grown on uncoated glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at RT and permeabilized with 0.1% Triton X-100 for 10 min at RT. After blocking nonspecific binding with 10% normal goat serum on 1% BSA (Abcam, Cambridge, UK), cells were incubated with antibody to a-SMA (ab32575; Abcam), rabbit IgG (NSC-2025; Santa Cruz Biotechnology, Santa Cruz, CA, USA), vinculin (V4139; Sigma) or Alexa 594-phalloidin (А12381; Molecular probe, Eugene, OR, USA) overnight at 4 °C. Antibody detection was performed using secondary antibodies conjugated with Alexa 488 (A11037; Invitrogen, Life Technologies, Grand Island, NY, USA) for 1 h at RT in the dark. The nuclei were counterstained with DAPI (D9542; Sigma,). Coverslips were mounted in AquaPolyMount (Polysciences Inc., Warrington, PA, USA). Images were captured using a Leica DM600B with a Leica DFC 420C camera (Leica Microsystems GmbH, Mannheim, Germany) and processed by FIJI software (GitHub Inc., Microsoft, Redmond, WA, USA).

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (Paneco, Moscow, Russia) for 10 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min. Non-specific binding was blocked with 10% normal goat serum (Abcam, Cambridge, UK) in 1% bovine serum albumin (Paneco, Moscow, Russia), and cells were stained with antibodies to alpha-actin (ab32575; Abcam; dilution 1/100), Alexa 594-phalloidin (A12381; Molecular probes, Eugene, OR, USA), or non-specific rabbit IgG (NSC-2025; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Staining with secondary antibodies conjugated to Alexa 488 or 594 (#A11034, #A21203, Invitrogen Waltham, MA, USA) was performed at room temperature in the dark for 1 h. The nuclei were stained with DAPI (D9542, Sigma-Aldrich, St. Louis, MO, USA). Images from at least 4 representative fields of view per well were obtained using an inverted microscope with a fluorescent module, Leica DMi8, and Leica DFC7000 T cameras (Leica Microsystems GmbH, Wetzlar, Germany), followed by processing with LasX (Leica Microsystems GmbH, Wetzlar, Germany) and FIJI (GitHub Inc., San Francisco, CA, USA).