Benchmark ultra ihc ish system

Manufactured by Roche

Sourced in United States, Switzerland

The BenchMark ULTRA IHC/ISH System is an automated slide staining platform designed for immunohistochemistry (IHC) and in situ hybridization (ISH) procedures. It automates the pre-treatment, staining, and detection steps, providing consistent and reliable results.

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Benchmark Ultra (524 citations) BenchMark ULTRA system (62 citations) BenchMark Ultra IHC/ISH staining system (8 citations) VENTANA BenchMark ULTRA IHC/ISH System (3 citations) BenchMark ULTRA Automated IHC/ISH slide staining system (3 citations) BenchMark ULTRA IHC system (2 citations)

18 protocols using benchmark ultra ihc ish system

Immunohistochemical and In Situ Hybridization Assessment of HER2 in Tumor Samples

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An IHC assay was performed for the detection of HER2 protein expression in FFPE tumor tissue specimens and cell lines. The FFPE tissue and cell line blocks were sectioned to a thickness of 4 to 5 μm and mounted on positively charged glass slides. Sections were dehydrated and heat-fixed in a laboratory oven at 60 °C for 1 hour to increase adherence of the tissue section to the glass slide. Sections were incubated with the PATHWAY anti-HER2/neu (4B5) rabbit monoclonal antibody (Ventana Medical Systems, Inc.), followed by incubation with a secondary antibody-horseradish peroxidase conjugate (ultraView Universal DAB Detection Kit, Ventana Medical Systems, Inc.) using the BenchMark ULTRA IHC/ISH System version 12.2 (Ventana Medical Systems, Inc.) per user instructions.
A dual ISH assay was performed to detect HER2 DNA amplification in FFPE tumor specimens that were IHC 2+. Sections were processed using the INFORM HER2 Dual ISH DNA Probe Cocktail, the ultraView SISH DNP Detection Kit, the ultraView Red ISH DIG Detection Kit (Ventana Medical Systems, Inc.), and accessory reagents using the automated BenchMark ULTRA IHC/ISH System version 12.2 (Ventana Medical Systems, Inc.) per user instructions.

Feng W., Inoue R., Kuwata T., Niikura N., Fujii S., Kumaki N., Honda K., Xu L.A., Goetz A., Gaule P., Cogswell J., Rimm D.L, & McGee R. (2023). Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens. Applied Immunohistochemistry & Molecular Morphology, 31(5), 339-345.

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Immunohistochemical Analysis of Lung and Heart Tissues

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Deparaffinized and rehydrated sections from the lung and heart were used for immunohistochemistry. Immunostaining was done with BenchMark Ultra IHC/ISH System fully automated instrument (Roche, Basel, Switzerland), using the ultraView Universal diaminobenzidine (DAB) Detection Kit (760–500, Roche-Ventana, Tucson, AZ 85755, USA), in accordance with the standard protocols supplied by the manufacturer. All antibodies were ready-to-use monoclonal antibodies (Ventana, Roche Diagnostics, Basel, Switzerland); antibodies directed against CD3 (Ventana, 2GV6) CD4 (Ventana, SP35), CD8 (Ventana SP57), CD20 (Ventana, LZ6), CD68 (Ventana KP-1), and CD45 (Ventana, LCA) were used. Negative control stainings were performed by omitting the primary antibodies.
All cases were independently analyzed by two pathologists without knowledge of the clinical diagnosis. The extent of immunoreactivity was assessed by using the same microscope at a magnification of 40×. The number of positive cells was quantified and expressed as cells/mm².

Colombo D., Del Nonno F., Marchioni L., Lalle E., Gallì P., Vaia F, & Falasca L. (2023). Autopsies Revealed Pathological Features of COVID-19 in Unvaccinated vs. Vaccinated Patients. Biomedicines, 11(2), 551.

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Immunohistochemical Profiling of MCC Samples

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Consecutive five-micrometer-thick tissue slides were prepared from formalin-fixed paraffin-embedded (FFPE) MCC patient samples for immunohistochemistry (IHC). IHC staining for CXCR4 started with deparaffinization followed by antigen retrieval in heated 1X citrate buffer (#H3300, Vector Laboratories) for 20min, blocking in normal goat serum (#S1012, Vector Laboratories), and overnight incubation with mouse monoclonal anti-human CXCR4 primary antibody (1:100, Clone 44716, #MAB172, R&D Systems). Slides were incubated with goat anti-mouse secondary antibody (#ab6789, Abcam) for 1 hour at room temperature and detected using a horseradish peroxidase detection system with DAB as chromogen (#SK4105, Vector Laboratories). IHC staining for ENO2 and vimentin was performed with the BenchMark ULTRA IHC/ISH system (Roche Diagnostics) using standard automated protocols. The primary antibodies ENO2 (pre-diluted, Clone MRQ-55, #760-4786, Ventana Medical Systems) and vimentin (pre-diluted, V9, 790-2917, Ventana Medical Systems) were incubated for 24minand 16min, respectively, and detected using a horseradish peroxidase detection (HRP) system with DAB as chromogen. Slides were then visualized, and images were captured using a stereomicroscope with a digital camera (Discovery V12 and AxioCam; Carl Zeiss, Inc).

Das B.K., Kannan A., Velasco G.J., Kunika M.D., Lambrecht N., Nguyen Q., Zhao H., Wu J, & Gao L. (2023). Single-cell dissection of Merkel cell carcinoma heterogeneity unveils transcriptomic plasticity and therapeutic vulnerabilities. Cell Reports Medicine, 4(7), 101101.

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Immunostaining and HPV Testing Protocol

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Immunostaining of p16^INK4a was performed with mouse monoclonal antibodies against the p16^INK4a antigen (clone E6H4; Roche, Basel, Switzerland), using the Optiview detection kit with the automated BenchMark ULTRA IHC/ISH system (Roche). p16^INK4A immunohistochemistry was scored positive when diffuse or block staining was observed and negative with a negative or patchy staining pattern.²²High‐risk HPV DNA‐testing was performed using the QIAscreen HPV PCR Test (Qiagen), as described previously for use on FFPE biopsy specimens.²³ The assay is directed against the E7 gene of 15 (probably) high‐risk HPV genotypes, that is, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67 and 68, with partial genotype information (HPV16 and −18).²⁴ Beta‐globin served as internal quality control. Samples were considered invalid for PCR testing when the cycle threshold (Ct) >30 for beta‐globin and no HPV was found.
HPV status was determined in all VIN and VSCC and not in controls. HPV status was considered positive when p16^INK4A and/or HPV PCR were positive, and negative when p16^INK4A was negative and HPV PCR was negative or invalid.

Thuijs N.B., Berkhof J., Özer M., Duin S., van Splunter A.P., Snoek B.C., Heideman D.A., van Beurden M., Steenbergen R.D, & Bleeker M.C. (2021). DNA methylation markers for cancer risk prediction of vulvar intraepithelial neoplasia. International Journal of Cancer, 148(10), 2481-2488.

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Immunohistochemical Staining of CD82 and S100A7

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CD82 and S100A7 single and dual staining and H&E staining were performed on a BenchMark ULTRA IHC/ISH System (Roche, Indianapolis, IN, USA). The paraffin-embedded tissue sections (4 μm) were de-paraffinized and incubated with CD82 and S100A7 antibodies (TS82b and MA1-91555), followed by Ventana Medical Systems ultraView Universal (or Optiview) DAB (3,3′-diaminobenzidine tetrahydrochloride) Detection Kit for CD82 and ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche Indianapolis, IN, USA) for S100A7. The slides were then counterstained with hematoxylin, mounted, and imaged using an Olympus BX51 microscope with an InfinityX camera and the Infinity analyze 3 6.5.5 software (Lumenera Corporation, Ottawa, ON, USA).

Reddi K.K., Zhang W., Shahrabi-Farahani S., Anderson K.M., Liu M., Kakhniashvili D., Wang X, & Zhang Y.H. (2024). Tetraspanin CD82 Correlates with and May Regulate S100A7 Expression in Oral Cancer. International Journal of Molecular Sciences, 25(5), 2659.

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Immunohistochemical Analysis of SSTR2 Expression in Cell Lines

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The immunohistochemical (IHC) staining was performed to verify the SSTR2 expressions in the cell lines HEK and HEKsst₂ with and without stimulation of epidrugs. The following combinations of 5-aza-dC/VPA concentrations were used, respectively: 0 µM/1.85 mM, 0.1 µM/1.85 mM, 3.9 µM/1.85 mM and 5.0 µM/1.85 mM. Each sample of HEK and HEKsst₂ cells was transferred into cell blocks. Cell blocks were cut into 1 µm sections, and automatically stained with hematoxylin-eosin staining (Dako Omnis, Aligent Technologies, Santa Clara, CA, USA). SSTR2 IHC staining was performed using the monoclonal antibody anti-SSTR2A (Zytomed Systems GmbH, Bargteheide, Germany) in a dilution of 1:25. The detection was realized by an OptiView DAB Kit (760–700, Roche Holding AG, Basel, Switzerland) using the secondary antibody cocktail, goat-anti-mouse and goat-anti-rabbit, following the manufacturer’s instructions. All above-described immunostainings were processed with the Benchmark ULTRA IHC/ISH System (Roche). Cell sections were covered by an automated cover-slipper (Klinipath, Duiven, Netherlands), digitalized and analyzed by the case viewer software 2.4 (Sysmex GmbH, Norderstedt, Germany). Positive staining of SSTR2 expressions was defined as a brown staining pattern of cell membranes.

Kotzerke J., Buesser D., Naumann A., Runge R., Huebinger L., Kliewer A., Freudenberg R, & Brogsitter C. (2022). Epigenetic-Like Stimulation of Receptor Expression in SSTR2 Transfected HEK293 Cells as a New Therapeutic Strategy. Cancers, 14(10), 2513.

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Immunohistochemical Analysis of GBM

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Serial unstained slides were cut from the formalin-fixed paraffin-embedded (FFPE) tissue blocks of GBM specimens from deceased, normal brain autopsy tissue from patients who died of non-neurological disease, and temporal lobectomy surgical specimens from patients with epilepsy with approved protocol from our Institutional Review Board. IHC for AR (clone SP107 rabbit monoclonal antibody, Cell Marque, Rocklin, CA, USA) was performed using BenchMark Ultra IHC/ISH system (Roche, Basel, Switzerland). Slides cut from the FFPE mouse brain GBM tissue were incubated with anti-AR (ab3510), anti-CD133 (ab19898), anti-Sox2 (ab97959) and anti-c-Myc (ab32072) individually or in combination. All these antibodies were from Abcam, Cambridge, MA, USA. After staining for the above markers with substrates incubated and color developed, slides were scanned with Ventana iScan HT slide scanner at 400× magnification and quantified using Definiens Tissue Studio (Ventana, Munich, Germany).

Zhao N., Wang F., Ahmed S., Liu K., Zhang C., Cathcart S.J., DiMaio D.J., Punsoni M., Guan B., Zhou P., Wang S., Batra S.K., Bronich T., Hei T.K., Lin C, & Zhang C. (2021). Androgen Receptor, Although Not a Specific Marker For, Is a Novel Target to Suppress Glioma Stem Cells as a Therapeutic Strategy for Glioblastoma. Frontiers in Oncology, 11, 616625.

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p16 IHC Staining Protocol

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IHC testing for p16 (E6H4, pre-dilute, Ventana) was repeated in our clinical laboratory only for the two cases with completely negative p16 IHC on initial testing. The block chosen for each case during initial p16 IHC was the same block utilized for repeat testing. The p16 IHC was applied to 4 μm thick sections taken from FFPE tissue blocks. The sections were placed in a BenchMark Ultra IHC/ISH System (Roche). Deparaffinization, pretreatment epitope retrieval, washing, blocking and incubation steps were all performed according to the manufacturer’s protocol. p16 staining was scored in an identical manner as described above.

Matson D.R., Accola M.A., Henderson L., Shao X., Frater-Rubsam L., Horner V.L., Rehrauer W.M., Weisman P, & Xu J. (2021). A “null” pattern of p16 immunostaining in endometrial serous carcinoma: An under-recognized and important aberrant staining pattern. International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists, 41(4), 378-388.

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Immunohistochemical Evaluation of Tumor Markers

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The BenchMark ULTRA IHC/ISH System (Ventana Medical Systems, Tucson, AZ, USA) was used to immunostain tissue samples for p27, p57, cyclin D1, nestin, and Ki-67 (Table 1). Positive and negative tissue controls were evaluated before interpreting any immunohistochemical results. For p27, p57, and cyclin D1, the percentage of tumor cells with stained nuclei was quantified. For nestin, the percentage of tumor cells with stained cytoplasm was quantified. The Ki-67 proliferation index was determined using the method of Wolfsberger et al. [23 (link)]. The percentages were enumerated within hotspots by counting at least 500 cells. Using these percentages, tumors were classified as either immunopositive or immunonegative based on a cutoff value. The cutoff value for both p27 and p57 was 10% (≥10% or <10%), and values <10% indicated immunonegativity or underexpression (mutant). For cyclin D1, the cutoff value was 50% (≥50% or <50%), and values ≥50% indicated immunopositivity or overexpression (mutant). For nestin, the cutoff value was 75% (≥75% or <75%), and values ≥75% indicated immunopositivity or overexpression (mutant). The cutoff value for the Ki-67 index was 17.5% (≥17.5% or <17.5%) based on the results of our previous study [7 (link)].

Iqneibi S., Nazzal J., Owda B., Sultan H., Amoudi R., Amarin J.Z., Al-Ghnimat S., Ahram M, & Al-Hussaini M. (2022). Immunohistochemical Expression of p27Kip1, p57Kip2, Cyclin D1, Nestin, and Ki-67 in Ependymoma. Brain Sciences, 12(2), 282.

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Immunohistochemical Analysis of CD30 and EBV

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Immunohistochemical analysis was performed using heat-induced epitope retrieval following standard procedures, employing antiCD30 (Ber-H2) mouse monoclonal primary antibody (G13408Z, Ventana Medical Systems Inc., Tucson, AZ, USA). The evaluation of EBV presence was performed using anti-human mouse Epstein–Barr virus/LMP1 monoclonal antibody (Clone CS1-4) (Master Diagnostica, Inc., Granada, Spain).
The detection of protein expression was performed with the automated immunostainer BenchMark ULTRA IHC/ISH System (Ventana Medical Systems Inc., Tucson, AZ, USA), following the manufacturer’s protocols.

Santisteban-Espejo A., Bernal-Florindo I., Montero-Pavon P., Perez-Requena J., Atienza-Cuevas L., Fernandez-Valle M.D., Villalba-Fernandez A, & Garcia-Rojo M. (2024). Pathogenic Variants Associated with Epigenetic Control and the NOTCH Pathway Are Frequent in Classic Hodgkin Lymphoma. International Journal of Molecular Sciences, 25(5), 2457.

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