Aquamount

Animals were anesthetized with a lethal dose of beuthanasia-D solution and transcardially perfused with 4% paraformaldehyde. Whole brains were removed, processed, embedded in paraffin, and cut into 4–6 μm sections. After de-parafinization, slides were incubated for 10 min at 95°C in Trilogy solution (Cell Marque), blocked with 3% hydrogen peroxide, followed by 2% normal goat serum. The sections were incubated with TBI or normal human serum overnight at 4°C, then incubated with goat anti-human HRP (Abcam) diluted in 2% goat serum. Staining was visualized with 3,3′-diaminobenzidine (DAB; Dako). The sections were counterstained with hematoxylin (Dako). Sections were finally washed with PBS, mounted, air-dried and cover slipped with Aquamount (Dako). The slides were scanned and examined using the Aperio ScanScope GL system at 20 x and ScanScope software. For immunofluorescence experiments, sections were incubated with anti-GFAP Alexa Fluor 555 conjugate (Cell Signaling) and either normal or TBI human serum overnight at 4°C, followed by goat anti-human Alexa Fluor 488 (Life Technologies) diluted in 2% goat serum. The sections were washed with PBS, mounted, air-dried and cover slipped with FluoroGel (GenTex). Staining was imaged with a Zeiss fluorescence microscope.

The presence of HPV, HSV-1, HSV-2 and CMV (Cytomegalovirus) in these tumors has been reported earlier [21 ]. Analyzed genotypes include both low risk (LR)-HPV-types 6/11/42/43/44/70 and high risk (HR)-types 16/18/30/31/33/35/39/45/51/52/53/56/58/59/66/68/73/82. The presence of EBV DNA was analyzed with an in-house PCR and Luminex xMAP-based method [22 ] using DNA extracted from the frozen tissue samples [22 ]. EBV gene target sequences: Forward primer: 5’-GAC-TGT-GTG-CAG-CTT-TGA-CGA-T-3, Reverse primer: 5’-CAG-CCC-CTT-CCA-CCA-TAG-GT-3’. Luminex probe: 5’-GGA-AAC-CAG-GGA-GGC-AAA-TCT-A-3’ [22 ].
The presence of EBV RNA transcripts EBER1 and EBER2 was examined with the “Epstein-Barr Virus (EBER) PNA Probe/Fluorescein and PNA-ISH Detection Kit” (Dako, Glostrup, Denmark) according to the manufacturer's instructions on the FFPE cancer samples. The substrate was incubated for 60 minutes, followed by a counterstain with Eosin and mounted using Aquamount (Dako). EBER-expression was graded from negative (−) to slight (+), moderate or intensive (+++), where most cells express EBER-RNA

Cells from the peritoneal lavage were sedimented by cytocentrifugation onto glass slides and fixed with 10% methanol-free formalin. Cells were stained with ORO for 30 min and counterstained with Mayer's Hematoxylin. Coverslips were mounted with Aquamount (Dako, Glostrup, Denmark). Microscopic images were taken using a Nikon Eclipse E600 microscope equipped with a Nikon Digital DS-U1 unit (Spach Optics Inc., New York, NY).