All tumors were classified according to the World Health Organization (WHO) classification of Tumours of Haematopoietic and Lymphoid Tissues version 2008 by conventional histological assessment on 2 µm hematoxylin and eosin (H&E) stained sections and on 4 µm immunohistochemically stained sections. Sections were cut from formalin‐fixed brain tumor tissues, embedded in paraffin blocks using standard pathology tissue processing procedures 20 . For immunohistochemistry, the following primary antibodies were used: CD3, CD5, CD10, CD19, CD20, CD79a, Bcl‐2, Bcl‐6, and Mib‐1. When appropriate this panel was extended with one or more of the following antibodies: BOB‐1, MUM1, CD 15, cyclin D1, Smlgkappa, Smlglambda, CD21, CD23, CD68, CD138, CD4, GFAP, CD31, CD43, TIA‐1, ALK‐1, CD8, and PAX‐5. All immunohistochemical procedures using primary and secondary antibodies and detection systems, were performed according to the manufacturer's recommendations on a Ventana Benchmark Ultra platform (Ventana Medical Systems Inc., Tucson, USA), tested and validated according to ISO 15189 standards. See Figure 1 for an example of a cerebral NHL, analyzed by histology with immunohistochemistry.
Ventana benchmark ultra platform
Manufactured by Roche
Sourced in United States
The Ventana Benchmark Ultra platform is a fully automated, high-throughput immunohistochemistry (IHC) and in situ hybridization (ISH) system designed for clinical diagnostic laboratories. It provides consistent and reproducible staining results by automating the entire staining process, from slide preparation to staining and coverslipping.
Automatically generated - may contain errors
Lab products found in correlation
Ck7 clone ov tl 12 30, by Agilent Technologies (1 mentions)
Ki 67 clone mib 1, by Agilent Technologies (1 mentions)
Cd56 clone 1b6, by Leica (1 mentions)
Chromogranin a, by Agilent Technologies (1 mentions)
Ez prep, by Roche (1 mentions)
Ck20 clone ks20, by Agilent Technologies (1 mentions)
Pl8 f6, by BioGenex (1 mentions)
M3619, by Agilent Technologies (1 mentions)
Optiview universal dab detection kit, by Roche (1 mentions)
Cd20 clone l26, by Roche (1 mentions)
Pd l1 clone 22c3, by Agilent Technologies (1 mentions)
Cd8 clone sp57, by Roche (1 mentions)
Cd4 clone sp35, by Roche (1 mentions)
Mrq 42, by Cell Marque (1 mentions)
Ultraview dab detection kit, by Roche (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Autostainer link 48 platform, by Agilent Technologies (1 mentions)
Ab7817, by Abcam (1 mentions)
Nanozoomer xr digital whole slide scanner, by Hamamatsu Photonics (1 mentions)
Ultraview universal dab detection kit, by Roche (1 mentions)
29 protocols using ventana benchmark ultra platform
1
Immunohistochemical Analysis of Brain Tumors
2
Immunohistochemical Profiling of Tumor Samples
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FFPE blocks were sectioned and mounted on coated slides (Dako, Glostrup, Denmark) and stained with H&E according to standard protocols. Tumor sections were deparaffinized using EZ-prep (Ventana Medical Systems®, Tucson, AZ, USA) and immunohistochemistry was performed using the Ventana Benchmark Ultra platform (Ventana Medical Systems) as previously described [33 (link)]. The following primary antibodies were employed: CD117 (polyclonal, 1:100, Dako (Glostrup, Denmark)), CD56 (clone 1B6, 1:50, Novocastra (Newcastle, UK)), chromogranin A (polyclonal, 1:2000, Dako), CK7 (clone OV-TL 12/30),1:1000, Dako), CK20 (clone KS20.8, 1:400, Dako), ki-67 (clone MIB1, 1:100, Dako), MYB (clone EP769Y 1:150 (AbCam, Cambridge, UK), napsin A (clone IP64, 1:400, Novocastra), and TTF-1(clone SPT24, 1:100, Novocastra). MYB was considered positive when nuclear staining was observed in at least 5% of tumor cells [31 (link)]. Positive controls as suggested on datasheets were used. Negative controls omitting the primary antibody were performed for all antibodies.
3
Immunohistochemical Analysis of Testicular Tissue
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The testicular tissue specimens were formalin‐fixed and paraffin‐embedded in cassettes and cut into serial sets of 2–3‐μm sections. Histological sections were stained with hematoxylin and eosin (HE) and incubated with immunohistochemical antibodies against D2‐40 (1:25, M3619, Dako, Glostrup, Denmark), CD99/MIC‐2 (1:100, 12E7, Dako), and placental‐like‐alkaline phosphatase (1200, PL8‐F6, Biogenex, Fremont, USA). Immunohistochemistry was performed automatically using the Ventana BenchMark ULTRA platform (Ventana Medical Systems, Tucson, USA) with positive and negative control tissue blocks per section for validation of each antibody. All specimens were validated and histopathologically reviewed by an experienced pathologist ECL. Included histological sections were stored as a separate research biobank for the purposes of these studies.
4
Immunohistochemical Profiling of CVID Gastric Cancer
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Serial 3-μm sections were prepared from one representative FFPE block. IHC staining was performed in all CVID and non-CVID gastric cancer cases with antibodies against CMV (clone CCH2 and DDG9, 1:1000; DAKO), CD20, (clone L26, prediluted; Ventana Medical Systems), CD8 (clone SP57, prediluted; Ventana Medical Systems), CD4 (clone SP35, prediluted; Ventana Medical Systems), GATA3 (clone D13C9, 1:200; Cell Signaling Technology), Foxp3 (clone AB54501, 1:100; abcam), CD138/Syndecan-1 (clone B-A38, prediluted; Cell Marque) and PD-L1 (clone 22C3, 1:80; DAKO). Samples were processed in the automatic Ventana Benchmark Ultra platform using an Optiview Universal DAB Detection Kit and an Optiview Amplification Kit for PD-L1 staining. EBV infection was studied using chromogenic ISH for EBV-encoded RNA (EBER-ISH, INFORM EBER probe, Ventana Medical Systems) using the same equipment, with enzymatic digestion (ISH protease) and an iViewBlue detection kit. The detailed protocols are presented in Table S1 .
5
Immunohistochemical Analysis of Mouse Tumor Specimens
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Mouse tumor specimens were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemistry was performed on representative 4 µm FFPE consecutive sections. Tissue sections were retrieved with EDTA buffer (pH 8) at 95° for 72 min. Slides were stained with rabbit monoclonal anti-human CD56 antibody (MRQ-42, Cell Marque, Sigma-Aldrich, 1:100) or mouse monoclonal anti-human CD45 antibody (2B11, PD7/26, Cell Marque, Sigma-Aldrich, 1:100) and developed on Ventana Bench-Mark ULTRA platform (Ventana-Roche, Tucson, USA) using the UltraView DAB detection Kit (Ventana-Roche). Slides were counterstained with haematoxylin.
6
PD-L1 Immunohistochemical Staining Protocol
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All specimens were fixed in 10% neutral buffered formalin fixative within 1 h of isolation. Fixation time was 6–72 h.Fifty samples were continuously sliced and at least five tissue sections were obtained per sample. HE staining and PD-L1 antibody staining were performed separately. Sufficient tumor tissues were identified on hematoxylin and eosin stained sections along with no less than 100 live tumor cells and their associated mesenchymal immune cells. These tissue sections were then stained for PD-L1. All sections stained with PD-L1 strictly following the manufacturer’s instructions on automatic immunohistochemistry. PD-L1 22C3 (Dako North America Inc, Carpinteria, CA) staining was performed using the Dako Autostainer Link48 platform; PD-L1 Ventana SP263 (Ventana Medical Systems, Tucson, USA), PD-L1 E1L3N (Aide Biomedical Technology Co., Ltd, Xiamen, China) and PD-L1 Ventana SP142 assay kit (Ventana Medical Systems, Tucson, USA) staining were performed using the Ventana Benchmark Ultra platform. All sections were scanned at 40X magnification on a UNIC digital pathology scanner (PRECICE 600 series), and their complete scanned images (WSI) were collected.
7
Rhesus Macaque Bone Marrow Biopsy Analysis
8
PD-L1 IHC Scoring in Tumors
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Serial sections of the TMAs were immunohistochemically stained for PD-L1 using the standardized 22C3 pharmDx assay on the Dako Autostainer Link 48 platform (Dako, Carpinteria, Ca) and the standardized SP263 and SP142 assays on the Ventana Benchmark Ultra platform (Ventana Medical Systems, Tucson, AZ), according to manufacturer’s instructions.
The stained slides were evaluated simultaneously and blindly by a dedicated urological pathologist with expertise in PD-L1 evaluation (EM) and by a researcher after appropriate training (MC); discrepancies between the two observers were resolved by consensus. All tissue cores have been evaluated using both CPS (combined positive score) and IC (tumor infiltrating immune cells) scoring systems: CPS is defined as the as the number of positive tumor cells, lymphocytes and macrophages, divided by the total number of viable tumor cells multiplied by 100 (at least 100 viable tumor cells), while IC is defined as the area of tumor infiltrated by PD-L1-stained immune cells divided by the total tumor area, multiplied by 100% (at least 50 viable tumor cells).
Necrotic areas and staining artifacts were excluded from scoring.
The stained slides were evaluated simultaneously and blindly by a dedicated urological pathologist with expertise in PD-L1 evaluation (EM) and by a researcher after appropriate training (MC); discrepancies between the two observers were resolved by consensus. All tissue cores have been evaluated using both CPS (combined positive score) and IC (tumor infiltrating immune cells) scoring systems: CPS is defined as the as the number of positive tumor cells, lymphocytes and macrophages, divided by the total number of viable tumor cells multiplied by 100 (at least 100 viable tumor cells), while IC is defined as the area of tumor infiltrated by PD-L1-stained immune cells divided by the total tumor area, multiplied by 100% (at least 50 viable tumor cells).
Necrotic areas and staining artifacts were excluded from scoring.
9
Immunohistochemical Staining of FFPE Tissues
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Single IHC staining was performed on 4 μm formalin-fixed paraffin-embedded tissue (FFPE) sections that were baked for 1 h at 60 °C. Slides were stained using the automated Ventana BenchMark ULTRA platform (Ventana, Roche diagnostic, Monza, Italy) and UltraView Universal DAB detection kit (Ventana, Monza, Italy). The sections were incubated with primary antibody for 1 hour using the mouse monoclonal antibody 15C4 against LRG1 [29 (link)] (dilution 1:100), the rabbit monoclonal EP373Y antibody against CD34 (Abcam, ab81289; dilution 1:100), and the mouse monoclonal antibody 1A4 against α-SMA (Abcam, ab7817; dilution 1:100). Immunohistochemistry (IHC) assay was carried out with appropriate positive and negative controls included in each staining run. The IHC stained sections were scanned and analyzed by a Hamamatsu NanoZoomer-XR digital whole slide scanner. Control eye specimens were resected from a patient with lacrimal sac tumor (male, 83 years) and from a patient with neuroblastoma (female, 3 years).
10
Automated Quantification of Tumor-Infiltrating Lymphocytes
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The primary antibodies and the antigen retrieval methods were described in Table S1 . Immunostaining with CD4 and CD8 was performed with the fully automated Ventana Benchmark ULTRA platform (Ventana) according to the manufacturer's instructions.
The microscopic images of CD4, CD8, and FOXP3 immunostaining were obtained using an objective lens of 20× with a NanoZoomer Digital Pathology system (Hamamatsu Photonics). The positive percentages of CD4, CD8, and FOXP3 at the periphery of tumor plus tumor‐liver interface were evaluated. The Automeasure function in Axio Vision 4.9.1 software (Carl Zeiss) was used to distinguish the immunopositive area and to calculate the occupied percentage of the immunopostive cells per tumor at the evaluated area. Positive stain was defined on the basis of the stained signal of the splenocyte as positive control.
The microscopic images of CD4, CD8, and FOXP3 immunostaining were obtained using an objective lens of 20× with a NanoZoomer Digital Pathology system (Hamamatsu Photonics). The positive percentages of CD4, CD8, and FOXP3 at the periphery of tumor plus tumor‐liver interface were evaluated. The Automeasure function in Axio Vision 4.9.1 software (Carl Zeiss) was used to distinguish the immunopositive area and to calculate the occupied percentage of the immunopostive cells per tumor at the evaluated area. Positive stain was defined on the basis of the stained signal of the splenocyte as positive control.
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