Rotifix

Tumor touch imprints or 5 µm frozen sections of primary tumors mounted on microscopy glass slides were fixed with 4% Rotifix (Roth) for 10 min at 4 °C and FISH was performed using a telomere PNA-Cy3 FISH probe (Dako/Life Technologies) according to the manufacturer’s instructions and a MYCN-digoxigenin (dig) probe (BAC clone RP11-355H10). Briefly, slides were pretreated with 0.05% pepsin in 0.01 N HCl for 1 min at 37 °C, followed by dehydration (70%, 96%, 100% EtOH series). Probes were applied, denaturation was carried out at 80 °C for 5 min and hybridization at room temperature for 3 h. After immersion in 1xPBS, slides were washed in standard saline citrate (SSC) containing 70% formamide at 42 °C for 15 min and twice with 0.1xSSC for 5 min. The MYCN-dig probe was detected using a sheep polyclonal anti-dig-FITC antibody (dilution 1:100; Roche 11207741910). Slides were washed in PNA Wash-buffer (Dako) and dehydrated in an EtOH series. Sections were counterstained and mounted with Vectashield containing DAPI (Vector laboratories) and covered with glass coverslips. Images were acquired using a Zeiss Axioplan2 microscope and the ISIS software version 5.7.4 (Metasystems).

Immunohistological analyses were performed on mouse brains harvested after cardiac perfusion with 1 × PBS. Samples were fixated in 4% Rotifix (Roth), 24 h, transferred into 30% sucrose in 1 × PBS, 24 h, RT and then frozen in TissueTek O.C.T. compound (Sakura) and stored at − 80 °C. 30 µm sagittal brain slices were obtained by cryotome sectioning (Frigomobil, Leica) and staining procedure was done in free-floating whole-brain sections. Brain slices were blocked 1 h, RT in 1 × PBS, 1% BSA, 0.2% Triton and 10% goat serum and incubated in primary antibody solution overnight at 4 °C. Samples were stained with monoclonal TBEV E antibody (MoAb19/1786) [22 (link)], monoclonal rabbit anti-GFAP (Sigma), monoclonal guinea pig anti-NeuN (Synaptic Systems), polyclonal rabbit anti-IBA-1 (Synaptic System) and polyclonal rabbit anti-cleaved caspase 3 [Asp175] (Cell Signaling Technologies) antibody. Subsequently, slices were incubated with DAPI (1 µg/mL), secondary anti-mouse, anti-guinea pig, or anti-rabbit antibodies conjugated with FITC, Cy3, or Cy5 (Jackson ImmunoResearch) for 1 h at RT. Representative pictures were taken by LSM5 (Zeiss) microscope at 20 × and 40 × magnification and are shown for each regions.

The lung tumor metastasis model was carried out as described previously [46 ]. A549 cells were studied for metastasis formation in severe combined immune deficient mice (NOD-SCID Il2rg^null, NSG) that were maintained under specific-pathogen-free conditions in the central animal facility of the University Hospital RWTH Aachen. All animal experiments were approved by local authorities in compliance with the German animal protection law (AZ 84–02.04.2013.A198). A549 cells were harvested, singularized, washed and resuspended in PBS. Subsequently, a uniform single cell suspension containing 3 × 10⁶ cells in 100 μl of PBS was intravenously injected into the lateral tail vein of 6–8 week-old SCID mice. After 35 days, lungs were prepared, fixed by intratracheal instillation of Roti-Fix^® (Roth, Germany), embedded in paraffin and cut in 3 μm slices. Hematoxylin-eosin staining was performed using standard protocols. Images were taken with a Zeiss microscope (AxioLab.A, Carl Zeiss MicroImaging GmbH, Germany) at 40-fold magnification. For mounting whole lung overviews the Keyence BZ-9000 software BZ2Viewer Merge was used. Tumor area was measured using ImageJ software 1.48v (Rasband, NIH, Bethesda, Maryland, US) and denoted as percentage of whole lung tissue area.