As previously described [21 (link)], 3-μm thick tissue sections were cut from formalin-fixed paraffin-embedded (FFPE) tissue microarray (TMA) blocks. After deparaffinization and rehydration with xylene and alcohol graded solutions, respectively, immunohistochemistry (IHC) was performed by using a Ventana Discovery XT Automated Slide Stainer (Ventana Medical System, Tucson, AZ, USA). Cell Conditioning 1 buffer (citrate buffer, pH 6.0; Ventana Medical System) was used for antigen retrieval. The slices were incubated with primary antibody against p53 (1:300, clone DO-7; Novocastra, Leica Biosystems, Newcastle Upon Tyne, UK), YAP1 (1:200, clone 63.7, Santa Cruz Biotechnology, Dallas, Texas, USA), ER (ER; 1:150, clone 6F11; Novocastra), PR (PR; 1:100; clone 16; Novocastra), HER2 (1:1500, DAKO, Glostrup, Denmark, clone polyclonal). The appropriate positive and negative controls were included.
Cell conditioning 1 buffer
Manufactured by Roche
Cell Conditioning 1 buffer is a laboratory reagent used to prepare cell samples for further analysis or processing. It is designed to maintain the integrity and functionality of cells during sample preparation steps. The core function of this buffer is to provide a controlled environment that supports the cells while they are being handled and processed.
Automatically generated - may contain errors
Lab products found in correlation
Ventana discovery xt automated slide stainer, by Roche (2 mentions)
Clone 16, by Leica Biosystems (1 mentions)
Clone do 7, by Leica Biosystems (1 mentions)
Clone 6f11, by Leica Biosystems (1 mentions)
Digital slide scanner, by Hamamatsu Photonics (1 mentions)
Bluing reagent, by Roche (1 mentions)
Flex ihc slides, by Agilent Technologies (1 mentions)
Discovery ultra autostainer, by Roche (1 mentions)
Hematoxylin 2, by Roche (1 mentions)
Ez prep solution, by Roche (1 mentions)
Optiview dab ihc detection kit, by Roche (1 mentions)
Benchmark xt, by Roche (1 mentions)
Iview dab detection kit, by Roche (1 mentions)
Anti idh1 r132h antibody h09, by Dianova (1 mentions)
Ventana optiview amplification kit, by Roche (1 mentions)
Hpa001906, by Merck Group (1 mentions)
Optiview hrp multimer, by Roche (1 mentions)
Optiview hq universal linker, by Roche (1 mentions)
6 protocols using cell conditioning 1 buffer
1
Immunohistochemical Analysis of Tumor Biomarkers
2
Multiplex Chromogenic IHC Panel Optimization
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A multiplex chromogenic immunohistochemistry panel (Figure 1 ) was implemented on the Discovery Ultra autostainer (Ventana Medical Systems, Tucson, AZ, USA) by sequential application of unconjugated primary antibodies with heat deactivation steps in between each sequence for elution purposes. Appropriate staining controls were performed during the panel development to check for possible cross‐reactivity related to the detection systems.
Three µm sections from formalin‐fixed, paraffin‐embedded tissue blocks were mounted on FLEX IHC slides (Dako, Glostrup, Denmark). The tissue sections were subjected to a standard immunostaining protocol including deparaffinization, epitope retrieval in Cell Conditioning 1 buffer (CC1, #950‐500, Ventana) for 32 min and blockade of endogenous peroxidase activity. Following the incubation and detection steps described in Table1 , the tissue slides were counterstained with Hematoxylin II (#790‐2208, Ventana) and Bluing Reagent (#760‐2037, Ventana), dehydrated and cleared. Coverslips were mounted using Pertex® Mounting Medium (#00811, Histolab Products AB, Gothenburg, Sweden). Slides were digitized using a Hamamatsu Digital Slide Scanner (Hamamatsu, Japan).
Three µm sections from formalin‐fixed, paraffin‐embedded tissue blocks were mounted on FLEX IHC slides (Dako, Glostrup, Denmark). The tissue sections were subjected to a standard immunostaining protocol including deparaffinization, epitope retrieval in Cell Conditioning 1 buffer (CC1, #950‐500, Ventana) for 32 min and blockade of endogenous peroxidase activity. Following the incubation and detection steps described in Table
3
Immunohistochemical Analysis of β-Catenin
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IHC for β-catenin was carried out on the 4 μm-thick sections of the TMA blocks. Briefly, sections of the paraffin blocks were deparaffinized using EZPrep solution (Ventana Medical Systems, Tucson, AZ), and heat-induced epitope retrieval was done under Cell Conditioning 1 buffer (Ventana). After blocking of the endogenous peroxidase under 3% hydrogen peroxide solution and washout, the sections were incubated with diluted anti-β-catenin primary antibody (titer 1:200; clone 14; Cell marque, Rocklin, CA) for 16 minutes. After the incubation with the secondary antibody, the antigen-antibody reaction signal was visualized under the OptiView DAB IHC Detection kit (Ventana). Finally, the sections were counterstained with hematoxylin. The TMA sections stained with β-catenin IHC were carefully reviewed by two pathologists (Y.K. and H.S.H.). Aberrant β-catenin expression was considered to be present when nuclear staining was apparent at any percentage within the examined tumor cells, regardless of cytoplasmic staining (S1 Fig ) [18 (link)]. Cytoplasmic staining of β-catenin was difficult to reliably discern from normal membranous staining in some circumstances and we therefore did not interpret cases with only cytoplasmic staining as having aberrant β-catenin expression.
4
Immunohistochemical Staining Protocol
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For immunohistochemistry, we used commercially available antibodies presented in Table 1 . Staining was performed in the BenchMark XT immunostainer (Ventana Medical Systems, Tuscon, AZ, USA). After antigen retrieval with Cell Conditioning 1 buffer (Ventana Medical Systems) at 95 °C for 30 min, the slides were incubated with primary antibodies against the antigens. The immunoreactivity was visualized using the iVIEW DAB Detection Kit (Ventana Medical Systems), according to the manufacturer’s instructions. Sections were counterstained with hematoxylin. Positive controls and negative controls omitting the primary antibodies were also included.
p16 immunohistochemistry was considered positive if strong and diffuse nuclear and cytoplasmic expression was found in at least 75% of the tumor [17 (link)].
PD-L1 immunohistochemistry was determined by using the combined positive score (CPS), defined as the number of PD-L1-staining cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen was considered to have PD-L1 expression if CPS ≥ 1 [18 (link)].
p16 immunohistochemistry was considered positive if strong and diffuse nuclear and cytoplasmic expression was found in at least 75% of the tumor [17 (link)].
PD-L1 immunohistochemistry was determined by using the combined positive score (CPS), defined as the number of PD-L1-staining cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells, multiplied by 100. The specimen was considered to have PD-L1 expression if CPS ≥ 1 [18 (link)].
5
Immunohistochemistry Protocol for Breast Cancer Biomarkers
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As described in previous studies [12 (link)], 3 µm thick tissue sections were cut from the TMA block. After deparaffinization and rehydration using xylene and alcohol graded solutions, respectively, immunohistochemistry (IHC) was performed using Ventana Discovery XT Automated Slide Stainer (Ventana Medical System, Tucson, AZ, USA). Cell conditioning 1 buffer (citrate buffer, pH 6.0; Ventana Medical System) was used for antigen retrieval. Appropriate positive and negative controls were included. Staining of all the IHC markers was assessed using light microscopy. A cut-off value of 1% nuclear staining or more was considered positive for ER and PR [13 (link)]. HER2 staining was interpreted based on the 2018 American Society of Clinical Oncology/College of American Pathologists guidelines [14 (link)]. Only strong and circumferential membranous HER2 expression (3+) was considered positive, while 0 and 1+ HER2 staining was considered negative. Cases with equivocal HER2 expression (2+) were further evaluated for HER-2 gene amplification using silver in situ hybridization.
6
Immunohistochemical Characterization of Gliomas
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Immunohistochemistry was conducted on 4 µm thick formalin-fixed, paraffin-embedded (FFPE) tissue sections mounted on StarFrost Advanced Adhesive slides (Engelbrecht, Kassel, Germany) followed by drying at 80°C for 15 min. Immunohistochemistry was performed on a BenchMark Ultra immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Sections were stained with anti-IDH1-R132H antibody H09 (Dianova, Hamburg, Germany) as previously described [3] . ATRX immunohistochemistry was performed as previously described [19] . In brief, after deparaffinization, slides were pretreated at 95°C in Cell Conditioning 1 buffer (Ventana) for 90 minutes. The sections were incubated with primary antibody HPA001906 (Sigma-Aldrich, St. Louis, MO; USA) diluted 1:200 for 2 hours. Standard Ventana signal amplification was used. For BRAFV600E staining V600E-specific clone VE1 was used. After pretreatment with cell conditioner 1 (pH 8) for 64 min, sections were incubated with VE1 hybridoma supernatant (monoclonal, dilution 1:5) at 37°C for 32 min. Antibody incubation was followed by OptiView HQ Universal Linker for 12 min, incubation with OptiView HRP Multimer for 12 min, signal amplification including the Ventana OptiView Amplification Kit (Ventana, catalogue number 760-099).
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