Cleaved caspase 3 clone 5a1e

The original tumor tissue and the explant of the tumor tissue were fixed in 10% neutral-buffered formalin and embedded in paraffin. Four microm sections were stained with H&E for the histological evaluation. For immunohistochemical analysis four micron paraffin sections were prepared, deparaffinized in xylene, and rehydrated with graded ethanol. Antigen retrieval was performed by microwave pretreatment. The following antibodies were employed for the analysis: Ki67 (clone 30-9, dilution, Ventana Medical Systems), p53 (clone DO-7, dilution 1:100, DAKO), p16ink4a (clone E6H4, dilution 1:50, MTM Laboratories), RB (clone 1F8, dilution 1:50, Thermoscientific), EGFR (clone 5B7 prediluted, Ventana Medical Systems) and cleaved caspase 3 (clone 5A1E, dilution 1:300, Cell Signaling). All stains were performed using the BenchMark XT Slide Preparation System (Ventana Medical Systems).

Cultured cells, fibers, and muscle sections were fixed with acetone or 4% formaldehyde solution and blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 5% goat serum. After blocking, they were stained with the following primary antibodies: anti-MyHC (clone MF20; eBioscience), cleaved caspase-3 (clone 5A1E; Cell Signaling Technology, Danvers, MA, USA), antilaminin-a2 (clone 4H8-2; Sigma), and anti-GFP (EMD Millipore, Billerica, MA, USA). After staining, they were incubated with a secondary antibody conjugated with Alexa-488 and -568 (Molecular Probes). After primary and secondary staining, EdU staining was performed using the Click-iT EdU Imaging Kit (Invitrogen), according to the manufacturer’s instructions. Nuclei were stained with 4,6′-diamidino-2-phenylindole (DAPI). Stained cells or sections were analyzed using a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). For mitochondrial analysis, MBs were incubated for 1 h with the MitoTracker® Deep Red FM (#8778; Cell Signaling), a dye that stains the mitochondria in live cells. Stained cells were analyzed using a confocal laser scanning microscope (SPF5; Leica).