Pepsin solution

Fluorescence in situ hybridisation (FISH) was performed on formalin-fixed, paraffin-embedded (FFPE) sections using a Zyto Light SPEC SMARCB1/22q12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany) and standard procedures. Sections were deparaffinised via descending ethanol series. For the pretreatment, the slides were placed in a steamer at 98 °C; citrate buffer (pH 6)/1 mM EDTA. The slides were digested with a pepsin solution (Zytomed Systems, Berlin, Germany) at 37 °C, pretreated with a saline–sodium–citrate solution at 37 °C and dehydrated in an ascending ethanol series. Dried, dehydrated slides were treated with the probe, denatured at 75 °C, and hybridised overnight at 37 °C. Slides were washed and counterstained with DAPI, then analysed with an Axio Imager.A2m microscope (Zeiss, Oberkochen, Germany), appropriate fluorescent filters, and imaging software (Ikaros Neon v.6.3.5, MetaSystems, Altlussheim, Germany). A total of 100 interphase nuclei were analysed for each specimen. A total of 15% of the nuclei demonstrating signal loss were defined as a cut-off for the deletion of the SMARCB1 gene locus.