IHC staining was conducted only in 79 patients, because in six cases, the paraffin-embedded blocks were insufficient. Pancytokeratin IHC staining was performed according to the instructions provided in the pathology laboratory manual. Briefly, we obtained unstained slide from paraffin-embedded tissue samples with 3 µm thickness. IHC staining was performed on an automated Ventana Benchmark XT platform (Roche Diagnostics, Basel, Switzerland) using the FDA-approved monoclonal mouse antibody AE1/AE3 clone with additional 4-fold dilution (PA0909, Leica Biosystems, Buffalo Grove, IL, USA) and a Ventana ultraVIEW DAB Detection Kit (Roche Diagnostics, Basel, Switzerland). IHC-stained ITBs (ITB-IHC) were counted using the four methods described above (Figure 2 ) (ITB-total, ITB-YN, ITB-ITBCC, and ITB×400).
Ventana ultraview dab detection kit
Manufactured by Roche
Sourced in United States
The Ventana Ultraview DAB detection kit is a lab equipment product designed for the detection of target molecules in tissue samples. The kit utilizes a diaminobenzidine (DAB) chromogen to produce a brown stain, which can be visualized under a microscope. The core function of this product is to enable the identification and localization of specific proteins or other biomolecules within a tissue sample.
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Lab products found in correlation
Ventana benchmark xt, by Roche (4 mentions)
Benchmark xt, by Roche (2 mentions)
Ventana benchmark xt processor, by Roche (1 mentions)
Ultraview universal alkaline phosphatase red detection kit, by Roche (1 mentions)
Ultraview universal dab detection kit, by Roche (1 mentions)
Alk antibody, by Cell Signaling Technology (1 mentions)
Cox 2, by Thermo Fisher Scientific (1 mentions)
Ck7 clone ov tl 12 30, by Agilent Technologies (1 mentions)
Ventana benchmark xt immunostainer, by Roche (1 mentions)
Clone cdx2 88, by Abcam (1 mentions)
Clone ks20, by Agilent Technologies (1 mentions)
9 protocols using ventana ultraview dab detection kit
1
Immunohistochemical Staining of Tumor-Infiltrating Cells
2
Tissue Microarray Immunohistochemistry Protocol
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Tissue microarrays were constructed for immunohistochemistry (IHC). Two tissue cores (3 mm) were obtained from two representative paraffin block-embedded tumor compartments (at the tumor margin and center; Figure 1 ). IHC staining was performed on an automated Ventana Benchmark XT platform (Roche Diagnostics, Basel, Switzerland) using monoclonal antibodies against CD15 (FDA-approved Ventana PATHWAY, MMA clone) and CD66b (1:100, 555723 [G10F5], BD biosciences, Franklin Lakes, NJ, USA), and the Ventana ultraVIEW DAB Detection Kit (Roche Diagnostics, Basel, Switzerland).
3
Immunohistochemical Staining of MATR3 in Tumor Tissues
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Immunohistochemical (IHC) staining was carried out on tissue macromatricescomposed of representative tumor areas. Five different large tissue fragments from donor paraffin blocks were present in one recipient block. The sections (4 μm thick) were cut from each tissue macroarray block and placed on high-adhesive glass slides (SuperFrost Plus; Menzel-Glaser, Braunschweig, Germany). The sections were subjected to IHC staining according to the previously described protocol [10 (link),11 (link)]. Immunohistochemistry was carried out by BenchMark® Ultra automatic staining machine (Roche Diagnostics/Ventana Medical Systems, Tucson, AZ, USA) with Ventana UltraView DAB Detection Kit (Ventana Medical Systems). MATR3 was detected by using anti-MATR3 rabbit polyclonal antibody (cat. no: HPA036565, Sigma-Aldrich, St. Louis, MO, USA) in a 1:300 dilution for 32 min.
4
Immunohistochemical Analysis of Breast Cancer Biomarkers
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Tissue sections were stained with Estrogen Receptor (SP1, CONFIRM), Progesterone Receptor (IE2, CONFIRM), and HER2 (4B5, PATHWAY). Immunohistochemistry was performed using the UltraView Universal DAB Detection Kit (Ventana). The immunohistochemistry of NIK (1:500, ab220442) and ALDH (1:500, 611195, BD) was performed using a Ventana Ultraview DAB detection kit (Ventana) and UltraView Universal Alkaline Phosphatase Red Detection Kit (Ventana), respectively. Diaminobenzidine or Fast Red was employed as the chromogen, and hematoxylin as the nuclear counterstain. All slides were processed in a Ventana BenchMark XT processor (Ventana, Tucson, AZ, USA). Slides were scanned in ScanScope CS2 (Aperio) and image analysis was conducted in ImageJ. This study has used paraffin embedded breast cancer tissue obtained from the Oncology Hospital in the XXI Century National Medical Center. All used tissues were part of residual material and they did not endanger patients diagnostic.
5
Immunohistochemical Staining for ALK Evaluation
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Immunohistochemical staining was performed on a Ventana BenchMark XT automated slide-processing system at Ventana Medical Systems. Immunohistochemical staining was performed using 2-μm sections of TMA blocks. All slides were stained with ALK antibody (clone D5F3 Cell Signaling Technology) diluted 1:50 using a Ventana Ultraview DAB detection kit in a Ventana BenchMark XT processor (Ventana Medical Systems, Inc, Tucson, AZ). Antigen retrieval was a standard automated process on the Ventana BenchMark XT at 37°C for 16 minutes. All immunohistochemical stains were evaluated independently by two pathologists. Specimens were scored 3+ if strong granular cytoplasmic brown staining in tumor cells was present (excluding non-tumor cells: alveolar macrophages, cells of neural origin, glandular epithelial staining), and the absence of cytoplasmic staining was classified a negative result as described previously [37 (link)].
6
IHC and SISH Evaluation of HER2 Expression
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The HER2 IHC-staining and HER2 SISH methods were performed following given instructions in the pathology laboratory manual. Briefly, for IHC analysis of HER2 expression, we selected a formalin-fixed, paraffin-embedded (FFPE) block containing both tumor and normal breast tissue to serve as an internal negative control. A HER2 SISH-positive breast cancer specimen was used as a positive control. The HER2 IHC staining was performed on an automated Ventana Benchmark XT platform using FDA-approved Ventana PATHWAY rabbit monoclonal antibody 4B5 clone and the Ventana ultraVIEW DAB Detection Kit (Ventana Medical Systems). HER2 expression was scored into one of four groups (0, 1+, 2+, 3+) according to the 2013 American Society of Clinical Oncology/College of American Pathologists Guideline Update1 (link): 0, no staining observed or membrane staining that is incomplete, faint, or barely perceptible in ≤10% of tumor cells; 1+, incomplete membrane staining that is faint or barely perceptible within >10% of tumor cells; 2+, circumferential membrane staining that is incomplete and/or weak/moderate and within >10% of tumor cells or with complete and circumferential membrane staining that is intense and within ≤10% of tumor cells; and 3+, circumferential membrane staining that is complete and intense within >10% of tumor cells.
7
Immunohistochemical Analysis of COX-2 in NMSC
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A total of 61 paraffin tissue archival blocks (4 μm thick, 10% neutral buffered formalin fixation) were immunohistochemically stained with COX-2 (1:100 diluted rabbit polyclonal antibody, Thermo Scientific). Primary antibody labeling was performed with an automated Ventana Ultraview DAB detection kit in a BenchMark XT (Ventana Medical Systems, Inc.). The study included 61 randomly selected incident cases with newly diagnosed NMSC, which was histologically confirmed. A minimum of 150 - 200 tumor cells were scored per patient (using × 40 objective). COX-2 immunohistochemical staining was considered positive if it was cytoplasmic or perinuclear.
The intensity of staining was scored as 0, 1, 2, or 3 as follows. 1) Score 0: no staining; 2) Score 1: weak/diffuse cytoplasmic staining (< 10% stained cells); 3) Score 2: moderate staining (10-90% stained cells); 4) Score 3: strong/intensive staining (> 90% stained cells). Low-grade adenocarcinoma of the colon was used as a positive external control for COX-2 staining.
The intensity of staining was scored as 0, 1, 2, or 3 as follows. 1) Score 0: no staining; 2) Score 1: weak/diffuse cytoplasmic staining (< 10% stained cells); 3) Score 2: moderate staining (10-90% stained cells); 4) Score 3: strong/intensive staining (> 90% stained cells). Low-grade adenocarcinoma of the colon was used as a positive external control for COX-2 staining.
8
Immunohistochemical Profiling of Tumor Markers
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Immunohistochemical (IHC) staining was performed on 5-μm-thick unstained sections from tissue microarray blocks using antibodies to cytokeratin 7 (CK7) (clone OV-TL12/30; Dako, Carpinteria, CA, USA; 1:300 dilution), to cytokeratin 20 (CK20) (clone Ks20.8; Dako; 1:200 dilution), to homeobox protein CDX2 (clone CDX2-88; Abcam, Cambridge, MA, USA; 1:100 dilution), and to epidermal growth factor receptor(EGFR) (clone 3C6; Roche Diagnostics, Mannheim, Germany; 1:100 dilution). IHC staining of human epidermal growth factor receptor 2 (HER2) was performed using the Ventana Ultra View DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA) and the Ventana PATHWAY HER2/neu rabbit monoclonal antibody (4B5) on a Ventana BenchMark XT immunostainer (Ventana Medical Systems). All slides from each tumor were evaluated by a single pathologist independently based on the following criteria. Expression of CK7, CK20, and CDX2 was considered positive if 10% of the tumor cells showed immunoreactivity. For EGFR, both the percentage of positive tumor cells and the intensity of positive staining were graded according to a previous report [23 (link)]. Total grades were generated on a scale of 0–6 and considered positive if the score was 2–6. The staining for HER2 was graded according to the guidelines for such testing in gastric cancers [24 (link)].
9
Immunohistochemical Staining of Formalin-Fixed Tissue
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The sections, 3-micron formalin-fixed, paraffin-embedded tissue, were deparaffinized and hydrated. Heat mediated antigen retrieval was done in citrate buffer pH 6.0 in a pressure cooker. Automated immunohistochemical staining was performed using a Ventana Benchmark XT automated stainer (Ventana Medical Systems, AZ, USA). The detection system was a Ventana Ultraview DAB detection kit (Ventana Medical Systems).
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