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6 protocols using aquamount

1

Immunohistochemical Analysis of TBI Samples

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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.
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2

Profiling Viral Infection in Tumor Samples

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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
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3

Lipid Droplet Staining and Microscopy

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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).
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4

Quantitative IHC Analysis of LXRβ

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Formalin-fixed, paraffin-embedded tumor sections were labelled using the ultraVIEW DAB system (Ventana, Basel, Switzerland). Briefly, antigen retrieval was carried out by heating slides for 90 minutes at 95°C in 1 mmol/L EDTA at pH 7.8. Slides were then incubated with LXRβ mAb (ab24361 (Abcam) at a 1/20 dilution for 32 min at 37°C. The stained arrays were counterstained with hematoxylin and mounted in Aquamount (Dako, Les Ulis, France). Labeling was detected using a Ventana Benchmark XT automat (Ventana).
Slides were then digitized using Hamamatsu NanoZoomer 2.0HT and NDP Scan software (Hamamatsu Photonics, Massy, France). Nucleus detection and quantification were performed using TissueStudio 3.6.1 software (Definiens, Munich, Germany).
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5

In Situ TUNEL Assay for Detecting DNA Fragmentation

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TUNEL staining was used to detect DNA fragmentation in situ and performed with the In Situ Cell Death Detection Kit, TMR red (Roche, Cat.-Nr. 12156792910) according to manufacturer's instructions. In brief, 40 μm brain sections of mouse brains were obtained as described above and blocked for 1 h at room temperature in TBS containing 0.5% Triton X-100, 0.1% Na-Azide, 0.1% Na-Citrate, and 5% normal goat serum. Sections were washed in PBS twice for 5 min each in PBS and incubated with the TUNEL labeling solution. Therefore, two brain sections were simultaneously incubated with 250 μl of TUNEL labeling solution in one well of a 24-well plate for 1 h at 37°C covered with aluminum foil. Sections were once washed with PBS containing Hoechst for 10 min at room temperature to stain nuclei. Before mounting sections in AquaMount (DAKO, Glostrup, Denmark) they were once washed in PBS for 10 min at room temperature. TUNEL positive (TUNEL+) cells were counted.
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6

EBV RNA Detection in Tumor Samples

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An in-house PCR and Luminex xMAP-based methods were used for EBV DNA detection as described in the previous studies [4 (link), 37 (link)]. Additionally, Epstein–Barr virus (EBV)-encoded small RNA (EBER) transcripts were examined by in situ hybridization (ISH) in tumor samples to ensure the latency of EBV. Due to tumor tissue unavailability, EBER ISH was available in 89.9% (142/158) of tumor samples. EBER PNA Probe/Fluorescein and PNA-ISH Detection Kit (Dako, Glostrup, Denmark) were used for detecting EBV RNA transcripts EBER1 and EBER2 from the TMA slides. The methodology has been described in more detail in a previous study [4 (link)]. The substrate was first incubated then treated with eosin and finally mounted in Aquamount (Dako). Tumor and stromal cells were evaluated and scored separately from TMA slides by two researchers (Reija Randén-Brady and Jaana Hagström). Inconclusive cases were rescored. A total of six punches of each tumor were scored; scoring results of EBER in TMA slides were defined as follows: negative (−), mild positivity (+), moderate positivity (++), and strong positivity (+++).
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