The status of EBV infection was assessed by an ISH detection for EBER on paraffin-embedded tissue sections using fluorescein-labeled oligonucleotide probes (INFROM EBER Probe, Ventana), as previously described [10 (link)]. The visualization system used was the Bench Mark XT with enzymatic digestion (ISH Protease 2, Ventana) and the iVIEW Blue v3 detection kit (Ventana). Appropriate positive and negative control sections were included for each run.
Ish protease 2
Manufactured by Roche
ISH Protease 2 is a laboratory product manufactured by Roche. It is a protease enzyme used for sample preparation in in-situ hybridization (ISH) techniques.
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Lab products found in correlation
4 protocols using ish protease 2
1
In Situ Detection of EBV
2
EBER Detection in Biopsy Samples
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Biopsy samples (4-μm thick) were mounted on glass slides to detect EBER using automated ISH (BENCHMARK, VENTANA MEDICAL SYSTEM, Oro Valley, AZ, USA), previously described21 (link). Briefly, tissue samples were treated with ISH protease 2 (catalog no. 780-4147; VENTANA MEDICAL SYSTEM) and labeled with an EBER-specific probe (catalog no. 780-2842; VENTANA MEDICAL SYSTEM), which was detected using the Inform Probes iView Blue V3 detection kit (VENTANA MEDICAL SYSTEM)21 (link). A sample was considered EBV (+) if definitive tumor cells were labeled with the EBER probe, and EBV (−) if the EBER-ISH was negative in tumor cells or positive only in bystander cells. The EBER staining patterns were interpreted by a hematopathologist who was blinded to the clinical outcomes of the patients. Histologic findings from a representative case is shown in Fig. 4 .![]()
Histologic examination of a monomorphic PTLD–DLBCL involving terminal ileum (
3
EBV Detection via EBER ISH in Tissue
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The status of EBV infection was assessed by ISH detection for EBER on paraffin-embedded tissue sections using fluorescein-labeled oligonucleotide probes (INFROM EBER Probe, Ventana), as previously described [11 (link)]. The visualization system used was the BenchMark XT with enzymatic digestion (ISH Protease 2, Ventana) and the iVIEW Blue v3 Detection Kit (Ventana). A known EBV-positive tumor was used as a positive control in each run. An appropriate negative control section was also included.
4
EBV Detection in Cancer Cells
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The presence of EBV in the cancer cells was assessed according to the protocol described in our previous study for EBV chromogenic in situ hybridization using an automatic staining device (Benchmark XT, Ventana, Tucson, AZ, USA) [11] . Briefly, 4-lm-thick sections were cut from representative blocks obtained from each patient, mounted onto coated slides, and dried at 74 °C for 30 min. After pretreatment with ISH protease 2 (Ventana) for 8 min at 37 °C, the slides were denatured at 85 °C for 12 min and hybridization was conducted at 57 °C for 1 h using EBV-encoded small RNA probes (INFORM, Ventana) that had been labeled with fluorescein. Detection was sequentially performed by applying mouse anti-fluorescein antibody and biotinylated goat antimouse antibody (iView blue; Ventana); counterstaining was performed using nuclear fast red. Strong staining in the nucleus was considered to indicate positivity, and the proportion of EBV-positive tumor cells was thereby evaluated.
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