Anti tropomyosin

LMS (n = 8) from one patient were used for histological analyses at various stages of cultivation (days: 0–2–5–10–14). LMS were washed in phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA), followed by paraffin embedding and sectioning at 7 µm. These sections were cut and mounted on coated slides and dried overnight at 37 ℃. Slides were dewaxed in xylene and hydrated using graded alcohols to tap water. The sections were consequently stained with haematoxylin and eosin (H&E; Sigma) for histological analysis and Sirius Red (Sigma) for detection of fibrillar collagen, and mounted using fluoromount (Southern Biotech). For immunofluorescence, sections were permeabilized with 0.1% Triton X-100 (Sigma), dissolved in 1% BSA in PBS for 10 min and blocked with 10% goat serum in PBS for 60 min. Then, the slides were incubated overnight with primary anti-Tropomyosin (1:250, Sigma T9283) diluted in 0.1% BSA in PBS at 4 °C. Secondary antibody incubation was performed at room temperature for one hour, followed by mounting with VECTASHIELD containing DAPI (Vectorlabs). Images were taken by a blinded investigator using the Zeiss LSM‐870 microscope.

Staining was performed as described^{28 (link),55 (link),56 (link)}. Primary antibodies: mouse monoclonal anti-mCherry (1:200, Clontech, 632543 or 1:50, DSHB, 3A11), anti-Tropomyosin (1:200, Sigma, T9283), anti-sarcomeric alpha Actinin (1:100, Abcam, ab9465), anti-Aurora B (1:200, BD Transduction Laboratories, 611083), anti-p27 (1:50, BD Transduction Laboratories, 610242), anti-Ki67 (1:250, Abcam, ab8191), rabbit polyclonal anti-Troponin I (sc-15368), anti-Cyclin A (sc-751), anti-cdc2 (sc-954), anti-Geminin (sc-13015) (all 1:50, Santa Cruz), anti-phospho-Histone H3 (Ser10) (1:200, Millipore, 06-570), anti-pRb807/811 (1:100, Cell Signaling, 9308), anti-mAG (1:300, MBL, PM011), anti p27 (1:50, SantaCruz, sc-528), anti-survivin (1:50, Novus, NB500-201K8), rat monoclonal anti-BrdU (1:100, Abcam, ab6326) and goat polyclonal anti c-kit (1:100, R&D Systems, AF1356). Immune complexes were detected with ALEXA 488- or ALEXA 594-conjugated secondary antibodies (1:200; Molecular Probes). DNA was visualized with DAPI (4′, 6′-diamidino-2-phenylindole, 0.5 g/ml). For BrdU, cells were cultured in 30 μM BrdU (neonatal: last 24 h, adult: last 5 days).

Staining was performed as described (24 (link)). Primary antibodies: anti-tropomyosin (1:200, Sigma), anti-actinin (1:100, Abcam), anti-aurora B (1:200) (both BD Transduction Laboratories), rabbit polyclonal anti-troponin I, anti-cyclin A, anti-cyclin dependent kinase 1 (cdc2), anti-geminin (all 1:50, Santa Cruz Biotechnology), anti-phospho-histone H3 (Ser10) (1:200, Millipore), anti-pRb807/811 (1:100, Cell Signaling), anti-mAG (1:300, MBL), rat monoclonal anti-5-Bromo-2-deoxyuridine (BrdU) (1:100, Abcam). Immune complexes were detected with ALEXA 488- or ALEXA 594-conjugated secondary antibodies (1:200; Molecular Probes). DNA was visualized with DAPI'(4” 6'-diamidino-2-phenylindole, 0.5 μg/ml). For BrdU, cells were cultured in 30 μM BrdU (Sigma) (neonatal: last 24–48 h, adult: last 5 days).