EBER-ISH was performed on the tissue sections using the BenchMark XT automated slide stainer (Ventana Medical Systems, Tucson, AZ, USA). The sections were treated with protease 2 (catalog no. 780-4147; Ventana Medical Systems) and labeled with an EBER probe (catalog no. 780-2842; Ventana Medical Systems) for 2 h. Hybridization products were visualized with Ventana ISH iView Blue Detection Kit (Ventana Medical Systems) according to the manufacturer’s instructions. Dark blue dots at the site of hybridization (nucleus) was regarded as positive for EBV.
Eber probe
Manufactured by Roche
The EBER probe is a laboratory equipment used for the detection of Epstein-Barr virus (EBV) encoded RNA (EBER) in clinical samples. The EBER probe is designed to hybridize with the EBER transcripts, which are abundantly expressed in EBV-infected cells. This probe can be used in various molecular diagnostic techniques, such as in situ hybridization, to identify the presence and localization of EBV infection in tissues or cell samples.
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Lab products found in correlation
Benchmark xt, by Roche (3 mentions)
Ventana ish iview blue detection kit, by Roche (3 mentions)
Protease 2, by Roche (1 mentions)
Iviewblue detection kit, by Roche (1 mentions)
Ish protease 3, by Roche (1 mentions)
Benchmark xt autostainer, by Roche (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Benchmark ultra, by Roche (1 mentions)
8 protocols using eber probe
1
Automated EBER-ISH for EBV Detection
2
In Situ Hybridization Protocol for EBV-encoded RNA
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In situ hybridization for EBV-encoded RNA was also performed on the BenchMark XT autostainer using fluorescein-conjugated oligonucleotide probes (EBER Probe, Ventana Medical System). In brief, 3-μm-thick sections from the tissue microarray blocks were deparaffinized and rehydrated, and then the sections were digested with a proteolytic enzyme (proteinase K at 37°C for 25 min). Thereafter, the slides were incubated with the probe at 55°C for 25 min and washed with a stringent solution. The slides were labeled with an anti-alkaline phosphatase-conjugated antibody to fluorescein. A chromogen (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium) was then added and counterstained with Mayer’s hematoxylin. Only cases with a strong signal within almost all tumor cell nuclei were considered positive (Figure 4E-4F ).
3
Chromogenic In Situ Hybridization for EBV
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Chromogenic ISH for EBV-encoded RNA (EBER) was performed in FFPE tissue samples with fluorescein-labelled oligonucleotide probes (EBER probe, Ventana) with enzymatic digestion (ISH protease 3, Ventana) and an iViewBlue detection kit (Ventana) with use of the BenchMark ULTRA staining system (K Tello).
4
Evaluating MMR Protein Expression and MSI Status
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MMR proteins expression was analyzed by IHC assay, using monoclonal antibodies directed against MutL homolog 1 (MLH1: VENTANA MLH1, M1), PMS homologue 2 (PSM2: VENTANA PMS2, EPR3947), mutS homologue 2 (MSH2: VENTANA MSH2, G219-1129) and mutS homologue 6 (MSH6: VENTANA MSH6, 44). The staining was regarded positive when the tumor nuclei stained positively with the same intensity of the internal positive control including infiltrating lymphocytes, stromal cells and adjacent non-neoplastic epithelium. All cases with loss (absence) of nuclear immunostaining or reduced protein expression, when compared with internal positive control, were considered negative. An abnormal MMR expression was represented by a patchy and/or weak expression consisting of a nuclear loss associated with a gain in cytoplasmic staining or a heterogeneous expression within adjacent tumor areas.15 (link)
Microsatellite instability status (MSI) molecular test will be performed by comparison of the allelic profiles of the mononucleotide repeat markers BAT-25, BAT-26, NR-21, NR-24, and NR-27 in tumor and corresponding normal tissue. EBER using VENTANA EBER Probe was performed to assess EBV status.
Microsatellite instability status (MSI) molecular test will be performed by comparison of the allelic profiles of the mononucleotide repeat markers BAT-25, BAT-26, NR-21, NR-24, and NR-27 in tumor and corresponding normal tissue. EBER using VENTANA EBER Probe was performed to assess EBV status.
5
In situ Hybridization for EBV Detection
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In situ hybridization for EBV was performed using the autostainer Bench Mark XT (Ventana Medical System, Tucson, AZ) with a Ventana ISH iVIEW Blue Detection Kit (Ventana Medical System) according to the manufacturer's instructions. Briefly, the prepared tissue sections were incubated with the EBER probe (800-2842, Ventana Medical Systems) and dark blue/purple at the site of the hybridization (nucleus) was interpreted as positive.
6
In Situ Hybridization for EBV
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EBV ISH was performed using the Bench Mark XT autostainer (Ventana Medical Systems, Tucson, AZ, USA) and Ventana ISH iVIEW Blue Detection Kit (Ventana Medical Systems), according to the manufacturer's instructions. Briefly, 4-µm-thick whole-slide sections, obtained with a microtome, were transferred onto silanized slides and allowed to dry for 10 min at room temperature, followed by 20 min in an incubator at 65 °C. The sections were treated with protease Z for 8 min, and incubated for 2 h with EBER probe (800-2842, Ventana Medical Systems) in the autoimmunostainer. Dark blue/purple at the site of hybridization (nucleus) was interpreted as positive for EBV (Fig. 1a, lower).
7
EBER-ISH Protocol for GC and PC
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We performed EBER‐ISH in FFPE tissues of GC and PC cases in our cohort using the VENTANA BenchMark ULTRA automated staining system (Roche) and an EBER probe (800–2842, Roche), as previously reported.33 We evaluated the stained slides using the Aperio ImageScope (Leica Biosystems Nussloch GmbH), and counted EBER‐ISH‐positive cells in 50 randomly selected 100‐μm2 fields.
8
Automated EBER Detection in Tissues
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To detect Epstein-Barr virus-encoded small RNAs (EBER), automated in-situ hybridisation was performed on VENTANA BenchMark ULTRA autostainer with Ventana EBER Probe and VENTANA ISH iview Blue Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany).
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