The tissue slides were deparaffinized in xylene, hydrated in alcohol, and baked in a microwave (30 min in Trisbuffer, pH 9). Endogenous peroxidase was blocked. Staining was performed on the Benchmark ultra-automated stainer (Ventana) using diamino-benzidine as chromogen (Dako, Glostrup, Denmark). The following antibodies were used: Pankeratin AE1/AE3 (clone PCK26, pre-diluted; Ventana Media Systems, Tucson, AZ ,USA); EMA (clone E29, pre-diluted; Ventana Media Systems, Tucson, Arizona ,USA); S100 protein (clone poly Z311, dilution 1:500; Dako, Glostrup, Denmark); smooth muscle actin (clone 1A4, dilution 1:12000; Sigma Aldrich, Saint Louis, MO, USA); desmin (clone DE-R-11, pre-diluted; Ventana Media Systems, Tucson, Arizona USA); myogenin (clone LO 26, dilution 1:20; Leica Biosystems, Buffalo Grove, IL, USA); MyoD1 (clone EP212, pre-diluted; Cell Marque, Rocklin, CA, USA); CD34 (clone QBEnd10, pre-diluted; Ventana, Tucson, AZ, USA); beta-catenin (clone 14, pre-diluted; Cell Marque, Rocklin, CA, USA); SOX10 (clone EP268, dilution 1:100; BioSB, Santa Barbara, CA, USA); H3K27Me3 (clone C36B11, dilution 1:200; Cell Signaling Technology, Danvers, MA, USA); PAX3 (clone 274212, dilution 1:100; RD Systems Europe, Lille, France)
For myogenin, MyoD1, SOX10, H3K27me3 beta-catenin and PAX3, only a nuclear staining was considered as positive.
For myogenin, MyoD1, SOX10, H3K27me3 beta-catenin and PAX3, only a nuclear staining was considered as positive.