Immunohistochemistry of Ewing Sarcoma

Formalin-fixed paraffin-embedded samples were collected at the Institute of Pathology of the LMU Munich^{57 (link)}. We harvested at least two cores per sample with a core-diameter of 1 mm from all blocks to construct tissue microarrays. All EwS samples showed cytogenetic evidence for a translocation of the EWSR1 gene either as determined by fluorescence in situ hybridization and/or qRT-PCR. The samples were reviewed by a reference pathologist. Four-micrometer sections were cut for immunohistochemistry and antigen retrieval was performed with microwave treatment using the antigen retrieval ProTaqs I Antigen-Enhancer (Quartett) for p-MYBL2 or the Target Retrieval Solution (Agilent Technologies) for cleaved caspase 3. In total, 7.5% aqueous H₂O₂ solution (room temperature) and blocking serum from the corresponding kits were used for 20 min for blockage of endogenous peroxidase. Then slides were incubated for 60 min with the primary antibodies anti-p-MYBL2 (1:100 dilution; Abcam, ab76009) and anti-cleaved caspase 3 (1:100 dilution, Cell Signaling, #9661). Afterwards slides were incubated with a secondary anti-rabbit IgG antibody (MP-7401, ImmPress Reagent Kit, Peroxidase-conjugated) followed by subsequent target detection using DAB+chromogen (Agilent Technologies). Slides were counterstained with hematoxylin Gill’s Formula (H-3401; Vector).