One million PF130 cells were inoculated in 200 μL serum free DMEM into the thoracic cavity of SCID female mice and waited 6 weeks for tumor formation. The weight of the animals was measured once a week. Then, the animals were divided into three groups (n = 9), control, trametinib (2 mg/kg) treated and MAP855 treated (15 mg/kg) and the drugs or the vehicle were administered and the animals weighed every Monday, Wednesday and Friday for three weeks. MAP855 and trametinib were dissolved in solvent containing 5% 1.0N HCl, 42% PEG300 and 20% solutol. At the end of the experiment, after opening up the chest, we thoroughly searched the whole thoracic cavity (including the surface of the pericardium and diaphragm) for tumor nodules and dissected them out. We pooled and weighed the nodules for each animal separately and fixed them in formalin. After embedding in paraffin, sections were stained with H&E as well as immunohistochemically labeled with ki67 (clon 30-9; monoclonal mouse IgG, Roche-Ventana) antibody following standard protocols on the Ventana BenchMark Ultra system (Roche Tissue Diagnostics, Grenzach-Vyhlen, Germany). The animal model protocol was performed following the Guidelines for Animal Experiments and was approved for the Department of Experimental Pharmacology in the National Institute of Oncology, Budapest, Hungary (PEI/001/2574-6/2015).
Ventana benchmark ultra system
Manufactured by Roche
Sourced in United States, Switzerland, Germany
The Ventana BenchMark ULTRA system is an automated slide staining platform designed for immunohistochemistry and in situ hybridization testing. The system automates the staining process, including deparaffinization, antigen retrieval, and the application of primary antibodies, detection reagents, and chromogens.
Automatically generated - may contain errors
Lab products found in correlation
Ab205921, by Abcam (4 mentions)
Virtuoso v 5, by Roche (3 mentions)
Rotary microtome, by Leica camera (3 mentions)
Antibody dilution buffer, by Roche (3 mentions)
Optiview dab ihc detection kit, by Roche (3 mentions)
Optiview dab system, by Roche (3 mentions)
D1m8i, by Cell Signaling Technology (2 mentions)
Goat anti rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Bond max, by Leica Biosystems (2 mentions)
Ventana optiview dab ihc detection kit, by Roche (2 mentions)
Leica suite version 2, by Leica Microsystems (2 mentions)
Ccp58, by Leica Biosystems (1 mentions)
Ultraview universal alkaline phosphatase red detection kit, by Roche (1 mentions)
Arcturusxt laser capture microdissection system, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by Nichirei Biosciences (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Cd4 4b12, by Nichirei Biosciences (1 mentions)
Il 1α, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Roche (1 mentions)
P16ink4a, by Roche (1 mentions)
48 protocols using ventana benchmark ultra system
1
Murine Xenograft Model of Lung Cancer
2
Comprehensive Immunohistochemical Analysis of Colorectal Neoplasms
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For immunostaining, an automatic and clinically validated instrument based on the Ventana Benchmark ULTRA system (Roche Tissue Diagnostics, Basel Switzerland) was used. Immunohistochemistry was performed with a new, enhanced-sensitivity, biotin-free multimer technology system, based on direct linkers between alkaline phosphatase and secondary antibodies (ultraView Universal Alkaline Phosphatase Red Detection Kit, Ventana Medical Systems, Basel Switzerland). In addition to oil red O staining, to assess lipid accumulation in the colorectal neoplasms, the primary antibody against adipophilin (1:50, AP125, Acris Antibodies, San Diego, CA, USA) was used. To classify phenotypic expression, the following primary antibodies were used: CD10 (1:20, 56C6, Leica Biosystems, Newcastle, UK), MUC2 (1:100, Ccp58, Leica Biosystems), CDX2 (1:50, CDX2-88, BIOCARE MEDICAL, Concord, CA, USA), MUC5AC (1:100, CLH2, Leica Biosystems), and MUC6 (1:100, CLH5, Leica Biosystems). Expression was classified as positive (negative) when more (fewer) than 5 % of the neoplastic cells in the neoplastic areas were stained.
3
PD-L1 Expression Quantification Protocol
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Specimens from 20 patients obtained between January 2000 and December 2013 were fixed with 10% buffered formalin. Formalin-fixed paraffin-embedded tissues were cut into 5 μm sections, deparaffinized, rehydrated, and stained in an automated system (Ventana Benchmark ULTRA system; Roche, Tucson, AZ, USA) using commercially available detection kits and antibodies against PD-L1 (28–8, ab205921; Abcam, Cambridge, MA, USA). PD-L1 was primarily localized to the cell membrane of tumor cells, and its expression was determined quantitatively by two pathologists based on the proportion of PD-L1-positive tumor cells. Cells were considered PD-L1-positive based on a ≥1% PD-L1 expression.
4
Molecular Profiling of Mixed Neuroendocrine-Acinar Carcinoma
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The sections were deparaffinized before the antigens were retrieved by heat treatment in an EDTA solution at pH 8.0. Protein expression was evaluated using 3-µm-thick formalin-fixed and paraffin-embedded (FFPE) sections with anti-chromogranin A (1:50 dilution; clone 5H7, NCL-CHROM-430; Novocastra, Newcastle, UK), anti-trypsin (1:400 dilution; Meridian Life Science, Memphis, TN, USA), anti-mucin 1 (MUC1) (1:50, clone DF-6; Novocastra), anti-Ki67 (ready-to-use, clone SP6, Nichirei, Tokyo, Japan), anti-TP53 (1:200, clone DO7; Leica Biosystems, Wetzlar, Germany) antibodies using using the Ventana BenchMark ULTRA system (Roche, Tucson, AZ, USA). A serial section of 10-µm FFPE tissue was stained with haematoxylin-eosin and then microdissected using an ArcturusXT laser-capture microdissection system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The ductal component and mixed NEC/ACC component were microdissected from FFPE tumor tissue. FFPE DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) (6 (link)).
5
Immunohistochemical Analysis of Tumor Samples
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IHC was performed following standard procedures. Briefly, tumor tissues were extracted, fixed in formalin, paraffin-embedded and subsequently sectioned in 5 µm coronal sections with a Leica rotary microtome. The following primary antibodies were used: Ki67 (Roche; Ref 790-4286, dilution 1:100), p53 (Roche; Ref 790-2912, predilution 1:20), IDH1 (vitro master diagnostica; Ref R132H antibody H09, predilution) and ATRX (Sigma-Aldrich; Ref HPA001906, 1:200). IHCs were analyzed by a senior pathologist of the Service and were performed following the manufacturer’s instructions on the Roche Ventana Benchmark ULTRA System with ethylenediaminetetraacetic acid (EDTA) pH 8.5 antigen retrieval. Sections were scanned with Virtuoso v.5.6.1 software (Ventana Medical Systems, Roche).
6
Immunohistochemical Analysis of Lung Surfactant Proteins
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Immunohistochemistry was performed with the Roche Ventana BenchMark Ultra System with Tris-EDTA (ABCA3) or citrate (proSP-C, SP-B, proSP-B) antigen retrieval using the following antibodies from Seven Hills Bioreagents at the indicated dilutions: ABCA3 (WRAB-ABCA3; 1:200); pro-SP-C (WRAB-9337; 1:2000); mature SP-B (WRAB-48604; 1:2000); and proSP-B (WRAB-55522; 1:800).
7
Immunohistochemical Analysis of PD-L1 and TILs
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Specimens from 20 patients obtained between January 2000 and December 2013 were fixed with 20% nonbuffered formalin, and those obtained from 19 patients between January 2014 and October 2017 were fixed with 10% buffered formalin (Table S1 ). Formalin-fixed paraffin-embedded tissues were sectioned at 5 μm, deparaffinized, rehydrated, and stained in an automated system (Ventana Benchmark ULTRA System; Roche, Tucson, AZ, USA) using commercially available detection kits and antibodies against PD-L1 (28–8, ab205921; Abcam, Tokyo, Japan), CD4 (4B12; Nichirei, Tokyo, Japan), CD8 (D1M8I; Cell Signaling Technology, Danvers, MA, USA), and CD3 (LN10; Leica Biosystems, Richmond, IL, USA). PD-L1 is primarily located in the cell membrane of tumor cells, and its expression was evaluated semi-quantitatively by two pathologists based on the proportion of PD-L1-positive tumor cells. A PD-L1 expression rate of 1% or greater was defined as PD-L1-positive. CD3 and CD8 were utilized as a marker for pan T lymphocytes and cytotoxic T lymphocytes, respectively. Thus, CD3+ and CD8+ lymphocytes were counted in the tumors, and the percentage of CD8+ lymphocytes in CD3+ lymphocytes was calculated as the TIL fraction, as previously reported [28 (link),29 (link),30 (link)].
8
Automated Immunohistochemistry Staining Protocol
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Formalin-fixed paraffin-embedded tissues were sectioned at 5 μm, deparaffinized, rehydrated, and stained in an automated system (Ventana Benchmark ULTRA system; Roche, Tucson, AZ, USA) using commercially available detection kits and 1:250 dilutions of antibodies against TTF-1 (SPT24; Biocare), PD-L1 (28–8) (ab205921; Abcam, Cambridge, UK), VTCN1 (D1M8I; Cell Signaling Technology, Danvers, MA, USA), and B7-H3 (D9M2L; Cell Signaling Technology). All slides were stained within two months post-sectioning. The cutoff for positive staining of TTF-1, B7-H3, and VTCN1 was >1% at any intensity, and samples were dichotomized as positive or negative. For PD-L1, expression was evaluated by two pathologists on a quantitative scale from 0% to 100%.
9
Formalin-Fixed Brain Tissue Analysis
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The animal brain tissues were fixed in 10% formalin. Then, the brain tissues were sliced into 5-μm-thick sections. Histological evaluation was performed by HE staining. The expression levels of p62 in the brain tissues were examined by IHC staining. IHC staining was performed by a Ventana BenchMark ULTRA system (Roche, Basel, Switzerland). The primary antibodies were diluted in Antibody Dilution Buffer (Ventana). Antigen retrieval was performed according to the standard protocol provided by the manufacturer. Secondary goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used. Apoptosis was evaluated by TUNEL staining according to the manufacturer’s protocol (Roche).
10
Glycophorin C Immunohistochemistry Protocol
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We used glycophorin C because this antibody has been routinely used in the automatic staining machine for immunohistochemistry of the diagnostic pathology laboratory of the University Medical Center Utrecht in hematopathology diagnostics. In this way we could guarantee that the staining was reproducible and has the same staining conditions in all slides of the study population. Although glycophorin A is more abundant with 5–9 × 105 copies, than the 0.5–1.0 × 105 copies of glycophorin C per cell, both give the same staining pattern of (remnants of) erythrocyte membranes due to the high prevalence of copies41 (link). To visualize glycophorin C, sections were stained with a monoclonal mouse anti-Glycophorin C antibody (clone Ret40f, DAKO, dilution 1:800) using the Ventana BenchMark Ultra system (Roche Diagnostics) with CC1 (EDTA) pretreatment during 24 min. The plaque characteristic glycophorin C was defined as the total area positively stained, adjusted for the total plaque size in μm2.
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