Cellb acquisition software

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).

Sections of 4 μm of representative tumor cryosections of resection specimens (Table 1) and of B-cell lymphoma control tissue were fixed in acetone at −20°C for 10 min (for IHC: supplemented with 0.3% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) to inactivate endogeneous peroxidase), followed by incubation in 10% normal goat serum (Dako, Glostrup, Denmark) in PBS buffer to block non-specific antibody binding.
Immunohistochemical expression of CD70 was assessed using the mouse monoclonal anti-CD70 2 F2 (IgG1, 0.16 μg/ml) antibody followed by a polyclonal goat anti-mouse/rabbit/rat IgG HRP-linker antibody conjugate (Brightvision, DPVO-110HRP; Immunologic, Duiven, the Netherlands) and DAB + Substrate Chromogen System (Dako) detection. All sections were examined with an Olympus BX41 microscope and Cell^B acquisition software (Olympus, Tokyo, Japan).
Immunofluorescent double-staining for CD3 and CD70 or CD3 and CD27 was performed with rabbit polyclonal anti-human CD3 (2.4 μg/ml; Dako), CD70 2 F2 and mouse monoclonal anti-human CD27 137B4 (IgG1, 1:200; Novocastra, Leica Microsystems, Wetzlar, Germany) followed by goat anti-rabbit Alexa 488 or goat anti-mouse IgG1 Alexa 546 (1:300; Invitrogen, Carlsbad, CA, USA). All sections were examined with a Leica DM5000 fluorescence microscope and LAS-AF acquisition program (Leica, Solms, Germany).