Tissue microarrays (TMA) were constructed as previously described [22 (link), 23 (link)], using a semi-automated arraying device (TMArrayer, Pathology Devices, Westminister, MD, USA). A set of three 1 mm cores was obtained from viable, non-necrotic areas of the primary tumours. For immunohistochemical (IHC) analysis of CD1a, CD68, CD163 and MARCO, 4 μm TMA-sections were pre- treated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc., Tucson, AZ, USA) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems Inc.) with the following antibodies: CD1a: clone NCL-CD1a-220, diluted 1:25, LEICA Biosystems, Newcastle, UK, CD68: clone KP1, diluted 1:1000, Dako, Glostrup, Denmark, CD163: clone 10D6 diluted 1:200 Novus Biologicals, Abingdon, United Kingdom, MARCO clone HPA063793, diluted 1:250, Atlas Antibodies, Bromma, Sweden. The antibody-antigen complex was visualized with ultraView Universal DAB Detection kit (Ventana Medical Systems Inc.).
Ventana benchmark stainer
Manufactured by Roche
Sourced in United States, Germany
The Ventana BenchMark stainer is a laboratory instrument used for the automated staining of tissue samples. It is designed to perform immunohistochemistry (IHC) and in situ hybridization (ISH) assays. The stainer automates the process of applying reagents, incubating, and washing the tissue samples to enable consistent and reproducible staining results.
Automatically generated - may contain errors
Lab products found in correlation
Ultraview universal dab detection kit, by Roche (2 mentions)
Anti cd3 antibody clone 2gv6, by Roche (2 mentions)
Autostainer plus, by Agilent Technologies (2 mentions)
Cd163 clone 10d6, by Novus Biologicals (1 mentions)
Clone 2gv6, by Roche (1 mentions)
Ultraview universal diaminobenzidine dab detection kit, by Roche (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Bond max stainer, by Leica (1 mentions)
Pd l1, by Cell Signaling Technology (1 mentions)
Nb100 40845, by Novus Biologicals (1 mentions)
Optiview dab ihc detection kit, by Roche (1 mentions)
Protease from streptomyces griseus, by Merck Group (1 mentions)
Basic dab detection kit 250 001, by Roche (1 mentions)
10 protocols using ventana benchmark stainer
1
Tissue Microarray Construction and Immunohistochemical Analysis
2
Immunohistochemical Profiling of Tumor Microenvironment
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Tissue microarrays (TMA) were constructed as previously described [13 (link), 14 (link)], using a semi-automated arraying device (TMArrayer, Pathology Devices, Westminister, MD, USA). A set of three 1 mm cores was obtained from viable areas of the 175 primary tumours. For immunohistochemical IHC analysis of CD56, CD3, CD68 and CD1A, 4 μm TMA-sections were pre-treated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc., Tucson, AZ, USA) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems Inc.) with the following antibodies: CD56: Clone MRQ-42, pre-diluted, Ventana Medical Systems Inc., CD3: Clone 2GV6, pre-diluted, Ventana Medical Systems Inc., CD1a: clone NCL-CD1a-220, diluted 1:25, LEICA Biosystems, Newcastle, UK, CD68: clone KP1, diluted 1:1000, Dako, Glostrup, Denmark. The antibody-antigen complex was visualized with ultraView Universal DAB Detection kit (Ventana Medical Systems Inc.).
3
Immunohistochemical Analysis of Tumor-Infiltrating Lymphocytes
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Tissue microarrays (TMAs) were constructed as previously described using a semiautomated arraying device (TMArrayer, Pathology Devices, Westminister, MD, USA) [26 (link)]. Duplicate tissue cores (1 mm) were obtained from two different blocks of the primary tumor.
For immunohistochemical analysis of FoxP3 and CD8, 4 μm TMA-sections were automatically pre-treated using the PT Link system and then stained in an Autostainer Plus (Dako; Glostrup, Denmark) with the anti-FoxP3 antibody (clone 236A/E7, mouse, dilution 1:200, Abcam, Cambridge, UK), and the anti-CD8 antibody (clone C8/144B, mouse; dilution, 1:50; product M7103; Dako). For immunohistochemical analysis of CD3, 4 μm TMA-sections were pretreated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc.Tucson, AZ, USA) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems Inc.) with the anti-CD3 antibody (clone 2GV6, pre-diluted, Ventana Medical Systems Inc).
Analysis of CD20 and IGKC was performed as previously described [33 (link)].
For immunohistochemical analysis of FoxP3 and CD8, 4 μm TMA-sections were automatically pre-treated using the PT Link system and then stained in an Autostainer Plus (Dako; Glostrup, Denmark) with the anti-FoxP3 antibody (clone 236A/E7, mouse, dilution 1:200, Abcam, Cambridge, UK), and the anti-CD8 antibody (clone C8/144B, mouse; dilution, 1:50; product M7103; Dako). For immunohistochemical analysis of CD3, 4 μm TMA-sections were pretreated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc.Tucson, AZ, USA) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems Inc.) with the anti-CD3 antibody (clone 2GV6, pre-diluted, Ventana Medical Systems Inc).
Analysis of CD20 and IGKC was performed as previously described [33 (link)].
4
Immunohistochemical Analysis of ER and PR
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For immunohistochemical analysis, 4 μm TMA-sections were automatically pre-treated using ULTRA Cell Condition Solution 1, pH 8.5 (Ventana Medical Systems, Tucson, AZ, USA) for heat induced epitope retrieval, and stained with the monoclonal antibodies CONFIRM anti-ER (SP1) and anti-PR (1E2) in a Ventana BenchMark stainer (Ventana Medical Systems). The antibody-antigen complex was visualized with UltraView Universal diaminobenzidine (DAB) Detection kit (Ventana Medical Systems).
Stained slides were digitalized at 20x magnification using the automated scanning system Hamamatsu, NanoZoomer, 13239-01, Hamamatsu Photonics, Sunayama-cho, Naka-ku, Hamamatsu City, Shizuoka, Japan). The immunohistochemical staining was manually annotated by three independent observers (GA, KJ and JEL) who were blinded to the clinical data and outcome of the patients, and with the two latter being board-certified pathologists, using the NDP.view2 viewer software version 3.2.12 (Hamamatsu Photonics). Expression of ER and PR was annotated as the number of tumor-associated stromal cells with nuclear staining. Conflicting annotations were jointly re-evaluated in order to reach consensus. The intensity of staining was not evaluated.
Stained slides were digitalized at 20x magnification using the automated scanning system Hamamatsu, NanoZoomer, 13239-01, Hamamatsu Photonics, Sunayama-cho, Naka-ku, Hamamatsu City, Shizuoka, Japan). The immunohistochemical staining was manually annotated by three independent observers (GA, KJ and JEL) who were blinded to the clinical data and outcome of the patients, and with the two latter being board-certified pathologists, using the NDP.view2 viewer software version 3.2.12 (Hamamatsu Photonics). Expression of ER and PR was annotated as the number of tumor-associated stromal cells with nuclear staining. Conflicting annotations were jointly re-evaluated in order to reach consensus. The intensity of staining was not evaluated.
5
Immunohistochemical Analysis of CD8, FoxP3, and CD3
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For immunohistochemical (IHC) analysis of CD8 and FoxP3, 4 μm TMA‐sections were pretreated using the PT Link system, and subsequently stained with the anti‐CD8 antibody (clone C8/144B, mouse; dilution, 1:50; product M7103; Dako) and the anti‐FoxP3 antibody (clone236A/E7, mouse, dilution 1:200, Abcam, Cambridge, UK) using the Autostainer Plus (Dako; Glostrup, Denmark).
For IHC analysis of CD3, 4 μm TMA‐sections were pretreated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc., Tucson, AZ) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems) with the anti‐CD3 antibody (clone 2GV6, prediluted, Ventana Medical Systems).
For IHC analysis of CD3, 4 μm TMA‐sections were pretreated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc., Tucson, AZ) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems) with the anti‐CD3 antibody (clone 2GV6, prediluted, Ventana Medical Systems).
6
Immunohistochemical Staining of CD57 in Prostate Cancer
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The immunohistochemical stains were performed with the avidin-biotin complex (ABC) method [11 (link)]. In the TMAs “incidental PCa,” ”prostatectomy 1,” “prostatectomy 2,” and “lymph nodes,” it worked on an automated stainer (Autostainer Link 48, DAKO, Denmark), using an anti-CD57 antibody (clone NK-1, Menarini, Berlin, Germany) at a dilution of 1 : 10 after a pretreatment with proteinase K for 10 min. For the TMA “prostatectomy 2,” immunohistochemical stains against prostate-specific antigen were already available from previous studies [12 (link)]. The TMA “prostatectomy 3” was stained on Ventana Benchmark stainer (Roche, Basel, Switzerland) with another CD57 antibody (clone TB01, DAKO) at a dilution of 1 : 50.
7
Immunohistochemical Analysis of KRAS and PIK3CA
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The KRAS antibody (clone 9.13, Thermo Fisher, dilution 1:100) and PIK3CA antibody (clone 6D9, Thermo Fisher, dilution 1:1000) stainings were performed on TMA slides using the Ventana Benchmark stainer (Roche Diagnostics, Germany) according to the protocol of the manufacturers. Expression of KRAS and PIK3CA in the cytoplasm of carcinoma cells were assessed according to the following criteria: negative or weak staining in <5% of tumor cells (score 0); weak staining ≥5–20% of tumor cells (score 1); moderate to strong staining in ≥20% (score 2; compare Figure 3 ). The evaluation of immunohistochemical expression was assessed manually by two pathologists (A.Q. and A.E.). Discrepant results, which occurred in a small number of samples, were resolved by consensus review. Additional immunohistochemical markers were evaluated in this cohort of EAC, parts of it (VISTA, CD3) published [39 ], others (PD-L1, LAG3, TIM3, IDO) are currently under review. In brief, monoclonal antibodies were used (Ventana: PD-L1; Cell Signaling Technology: LAG3, TIM3, IDO) on the Ventana Benchmark stainer (PD-L1) or the Leica BOND-MAX stainer (Leica Biosystems, Germany) (LAG3, TIM3, and IDO). The expression in <1% lymphocytes were defined as negative and ≥1% was assessed as positive.
8
Immunohistochemical Analysis of DOT1L
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Immunohistochemistry (IHC) was performed on TMA slides using the primary rabbit polyclonal antibody specific for DOT1L (#NB100-40845; dilution, 1:50; Novus Biologicals, Ltd., Cambridge, UK). Immunohistochemical staining was performed using the Ventana BenchMark stainer (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol with on-slide controls of the appendix. As a secondary antibody the ready-to-use Ventana Detection kit (cat. no. 760–700; OptiView DAB IHC Detection kit; Ventana, Roche Diagnostics GmbH, Mannheim, Germany) for indirect biotin-free detection of primary rabbit antibody was used. DOTL1 exhibits a nuclear staining pattern. DOT1L staining was evaluated manually using light microscopy by two pathologists and discrepant results were resolved by consensus review.
9
Immunohistochemical Staining of Renal Tissue
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Immunohistochemical stainings with antibodies specific for IgA, IgG, IgM, C1q, C3c (all polyclonal: Cat. No. IgA A0262, IgG A0423, RRID:AB_2335700 ; IgM A04202, RRID:AB_578520 ; C1q A013602, RRID:AB_578496 ; C3c A006202, RRID:AB_578477 , Agilent, Santa Clara, CA, USA) were performed on formalin fixed and paraffin-embedded (FFPE, 1μm sections) material with current standard methods after digestion with protease from Streptomyces griseus (Sigma-Aldrich, Munich, Germany, P5147) on a Ventana Benchmark stainer (Roche, Basel, Switzerland) or manually before 2011. Limited staining in the glomerular vascular pole was scored as negative. The intensity of staining was categorized into 4 grades: grade 0 (none), grade 1 (mild), grade 2 (moderate), grade 3 (strong).
In case of IgA reactivity in the time zero biopsy additional stainings for C4d (polyclonal, rabbit anti-C4d, 1:500, Cat. No. RBK061,RRID:AB_2864450 , Zytomed Systems GmbH, Bargteheide, Germany; antigen-retrieval with ULTRA CC1 buffer, Roche) and galactose-deficient IgA (rat anti-Gd-IgA1, clone KM55, 1:100, Cat. No. 10777, Immuno-Biological Laboratories, Minneapolis, MA, USA; antigen-retrieval with protease digestion) were performed manually.
In case of IgA reactivity in the time zero biopsy additional stainings for C4d (polyclonal, rabbit anti-C4d, 1:500, Cat. No. RBK061,
10
Comprehensive Post-Mortem Examination Protocol
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A post-mortem examination was performed on patient KEN-334-6, who signed an informed consent form, for the French National Brain Bank Network Donation Program NeuroCEB.
The postmortem delay was 11 h. The right half of the brain, including the hemisphere, brainstem, and cerebellum, was fixed by immersion in 4% formaldehyde (10% formalin). The contralateral hemisphere was frozen at -80°C. Formalin-fixed brain samples from selected regions, including the cerebral cortex, hippocampus, basal ganglia, cerebellum, brainstem, spinal cord, and anterior and posterior roots of the spinal nerves, were embedded in paraffin and cut at a thickness of 3 μm. The spinal cord was sampled at the cervical, thoracic, and lumbar levels. The sections were deparaffinized in graded alcohol solutions and stained with hematoxylin-eosin (HE) and HE combined with Luxol fast blue for myelin. Selected sections were immunostained using a Ventana BenchMark stainer (Roche TM , Tucson, AZ, USA). The biotinylated secondary antibody was included in the detection kit (Ventana Medical Systems Basic DAB Detection Kit 250-001). Diaminobenzidine (DAB) was used as a chromogen. The pretreatment and antibodies used for immunohistochemistry are listed in Supplemental Table S2. Genomic DNA was isolated from cerebellum, according to standard procedures.
The postmortem delay was 11 h. The right half of the brain, including the hemisphere, brainstem, and cerebellum, was fixed by immersion in 4% formaldehyde (10% formalin). The contralateral hemisphere was frozen at -80°C. Formalin-fixed brain samples from selected regions, including the cerebral cortex, hippocampus, basal ganglia, cerebellum, brainstem, spinal cord, and anterior and posterior roots of the spinal nerves, were embedded in paraffin and cut at a thickness of 3 μm. The spinal cord was sampled at the cervical, thoracic, and lumbar levels. The sections were deparaffinized in graded alcohol solutions and stained with hematoxylin-eosin (HE) and HE combined with Luxol fast blue for myelin. Selected sections were immunostained using a Ventana BenchMark stainer (Roche TM , Tucson, AZ, USA). The biotinylated secondary antibody was included in the detection kit (Ventana Medical Systems Basic DAB Detection Kit 250-001). Diaminobenzidine (DAB) was used as a chromogen. The pretreatment and antibodies used for immunohistochemistry are listed in Supplemental Table S2. Genomic DNA was isolated from cerebellum, according to standard procedures.
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