Swine serum

Snap frozen tumors were sectioned on a cryostat and mounted on slides. Slides were fixed for 30 min using −20 °C cold methanol. Slides were incubated in a humidified chamber at 37 °C for 30 min with swine serum (Dako, Glostrup, Denmark). The serum was removed and the slides were incubated with anti-ANXA2 (dil 1:500) and anti-PRDX2 (dil 1:500) for 1 h in a humidified chamber at 37 °C. After washing three times with PBS for 10 min at RT, slides were incubated with the appropriate FITC-conjugated secondary antibodies in a humidified chamber at 37 °C for 1 h. After washing as described above, chamber slides were mounted on glass cover slips using Vector mounting solution for fluorescence (Vector Laboratories, UK) and viewed using a Zeiss LSM 510 META -Laser Scanning Confocal Microscope (Carl Zeiss Inc., Oberkochen, Germany.

Biopsies were taken at diagnosis from eight patients with ERMS. Four-μm sections containing representative tumor, as verified by an independent pathologist, were deparaffinized, and citrate antigen retrieval and endogenous peroxidases inactivation were performed. RMS cells in the biopsy sections were discerned by staining for myogenin (MYF4; #L026; Immunologic, Duiven, the Netherlands). Expression of the following ligands was assessed using rabbit polyclonal antibodies overnight at 4 °C: CD155 (hpa012568), ULBP-1 (hpa007547) and CD112 (hpa012759; all Sigma-Aldrich; Zwijndrecht, the Netherlands). MICA expression was assessed using a goat polyclonal antibody anti-MICA (AF1300, R&D Systems, Oxon, United Kingdom) overnight at room temperature on sections pre-treated with 10 % swine serum (Dako, Heverlee, Belgium) for 30 min at room temperature.
Antibody binding was detected by Liquid DAB + Substrate Chromogen System (Dako, Heverlee, Belgium) after applying Dako Envision + System–HRP-labeled Polymer Anti-Rabbit for the rabbit polyclonals or Dako LSAB + System–HRP for the goat polyclonal-treated sections. Testis was used as an internal positive control for the activating NK ligands. All samples were counterstained with hematoxylin, mounted with Pertex, and examined under a light microscope using CellB acquisition software (Olympus, Zoeterwoude, the Netherlands).