Vectafluor amplified kit

After the indicated treatments, SkMs were seeded on chamber slides (3500 cells/cm²). After 24 h, the supernatant was removed, and the cells were washed with PBS (PAN Biotech, Aidenbach, Germany), covered with 4% formaldehyde solution (27248, Otto Fischar GmbH, Saarbrücken, Germany) for 10 min and rinsed with 0.1% Tween 20 (9127.1, Carl Roth GmbH, Karlsruhe, Germany) in PBS 3 times. Fixed SkMs were permeabilized for 10 min using 0.1% Triton X-100 (T8787, Merck KGaA, Darmstadt, Deutschland) in PBS and incubated with 1:200 diluted skeletal muscle actin antibody (MA5-12542, Thermo Fisher Scientific, Waltham, MA, USA) and the VectaFluor Amplified Kit (DK2488, Vector Laboratories, Burlingame, CA, USA) or with 1:200 diluted desmin antibody (ab227651, Abcam, Cambridge, UK) and the VectaFluor Amplified Kit (DK1594, Vector Laboratories) for staining, according to the manufacturer’s protocol. After staining, cells were covered with a coverslip in mounting medium with DAPI (DK2488, Vector Laboratories).