Immunohistochemistry was performed on fresh 5-μm sections from FFPE blocks on Leica BOND-MAX (Leica Biosystems, Buffalo Grove, IL) automated staining system. Briefly, slides were deparaffinized and subjected to antigen retrieval in a pH = 9 buffer. Primary antibodies (ALK – clone 5A4 – Abcam, Cambridge, UK, 1:50 dilution; ROS-1 – clone D4D6 – Genemed Biotechnologies, San Francisco, CA, 1:100 dilution; PD-L1 – clone E1L3N – Cell Signaling Technology, Danvers, MA, 1:400 dilution) were incubated for 60, 60 and 20 min respectively. Revelation was performed with Leica BOND-MAX detection kits. ALK and ROS1 results were interpreted as positive or negative. PD-L1 result was expressed as the percentage of stained tumor cells.
Bond max
Manufactured by Leica
Sourced in Germany, United States, United Kingdom, Australia, Japan, Italy, Switzerland, Israel, Canada, France, Sweden, Singapore
The Bond Max is a laboratory instrument used for automated in-situ hybridization (ISH) and immunohistochemistry (IHC) staining. It is designed to streamline and standardize these processes, providing consistent and reliable results. The Bond Max handles sample preparation, staining, and slide processing, allowing for efficient high-throughput analysis of tissue samples.
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Lab products found in correlation
Bond polymer refine detection kit, by Leica (78 mentions)
Bond polymer refine detection, by Leica (17 mentions)
Bond epitope retrieval solution 2, by Leica (17 mentions)
Bond epitope retrieval solution 1, by Leica (12 mentions)
Scn400 slide scanner, by Leica (11 mentions)
Bond polymer refine detection system, by Leica (11 mentions)
Bond dewax solution, by Leica (9 mentions)
Bond primary antibody diluent, by Leica (9 mentions)
Iview dab detection kit, by Roche (9 mentions)
Refine detection kit, by Leica (9 mentions)
Ventana benchmark xt, by Roche (7 mentions)
Aperio cs o slide scanner, by Leica (6 mentions)
Polymer refine detection kit, by Leica (6 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (6 mentions)
Benchmark xt, by Roche (5 mentions)
Bond refine polymer staining kit, by Leica (5 mentions)
Bond wash solution, by Leica (5 mentions)
Epitope retrieval solution 1, by Leica (5 mentions)
Bond 3, by Leica (4 mentions)
Pd l1 clone e1l3n, by Cell Signaling Technology (4 mentions)
596 protocols using bond max
1
Immunohistochemical Analysis of Lung Cancer Biomarkers
2
Immunohistochemical Analysis of AQP 5 and p-NF-κB in Lung Tissue
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Immunohistochemistry was performed to detect AQP 5 and p-NF-κB expression in lung tissues. AQP 5 is a specific transmembrane protein responsible for water transport and is expressed in type I lung epithelial cells (13 (link)). An automatic immunohistochemistry system, Leica BOND-MAX, was used in this study. Rabbit polyclonal antibodies against AQP 5 (ab104751; Abcam, USA) and p-NF-κB (3033; Cell Signaling, USA) were used at a dilution of 1:1000 and stained with the Leica BOND-MAX system. A DAB enhancer (Leica Microsytems, UK) was applied to increase the intensity of DAB staining. Finally, the samples were analyzed with light microscopy.
3
Xenograft Tumor IHC Staining
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4
Immunohistochemical Analysis of Tumor Markers
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Tissue sections were prepared by embedding fixed tumor tissue in paraffin, then dewaxing and rehydrating them. Antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0 or 9.0) at 98 °C for 20 min. Immunohistochemical staining (IHC) was performed on the tissue sections using an automatic tissue processor (Bond-Max; Leica Microsystems, Wetzlar, Germany) according to the standard protocol. Briefly, the sections were incubated with primary antibodies anti-E-cadherin (Proteintech), anti-phospho-Erk1/2 (Biolegend), and anti-cleaved-Caspase 3 (CST). The sections were then treated with HRP-conjugated secondary antibody (BOND Polymer Refine Detection (Leica)) according to the standard protocols of the instrument after washing. The tissue sections were stained with diaminobenzidine and counterstained with hematoxylin. The resulting tissue slides were examined using a BZ-X710 microscope (Keyence, Osaka, Japan).
5
Immunohistochemical Evaluation of TP53 and MDM2 Protein Expression
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The same tissue block used for extracting genomic DNA was selected from each case slide. Serial 3-μm thick sections were made from FFPE tissue blocks.
Tissue sections were deparaffinized and rehydrated. After heat-induced antigen retrieval, IHC was performed using an automated immunostainer (Bond-Max, Leica MicroSystems, Wetzlar, Germany) with monoclonal antibodies against TP53 (clone DO7; dilution, 1:200) and MDM2 (clone 1B10; 1:200). Nuclei were counterstained with hematoxylin. Nuclear staining of TP53 and MDM2 protein was shown as brown granules. A positive result was defined as more than 30% of the tumor cells showing positive staining.
All slides were reviewed in a blinded manner without clinical information by two independent authors (H. I. and K.M.). When the assessment was different between the two observers, agreement was reached using a double-headed microscope. These slides were observed using a light microscope (Nikon ECLIPSE 80i, Nikon Corporation Tokyo, Japan) with a 40× field objective and 10× ocular lens corresponding to a field diameter of 550 μm for the slides.
Tissue sections were deparaffinized and rehydrated. After heat-induced antigen retrieval, IHC was performed using an automated immunostainer (Bond-Max, Leica MicroSystems, Wetzlar, Germany) with monoclonal antibodies against TP53 (clone DO7; dilution, 1:200) and MDM2 (clone 1B10; 1:200). Nuclei were counterstained with hematoxylin. Nuclear staining of TP53 and MDM2 protein was shown as brown granules. A positive result was defined as more than 30% of the tumor cells showing positive staining.
All slides were reviewed in a blinded manner without clinical information by two independent authors (H. I. and K.M.). When the assessment was different between the two observers, agreement was reached using a double-headed microscope. These slides were observed using a light microscope (Nikon ECLIPSE 80i, Nikon Corporation Tokyo, Japan) with a 40× field objective and 10× ocular lens corresponding to a field diameter of 550 μm for the slides.
6
Immunohistochemistry for MMR Proteins
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IHC was carried out in cores from 2 μm thick TMA sections with the following antibodies and conditions: MLH1, clone ES05 (Monosan, Uden, Netherlands) at 1:80 dilution; MSH2, clone 25D12 (Novocastra/Leica Microsystems, Wetzlar, Germany) at 1:40 dilution; MSH6, clone EP49 (DAKO, Glostrup, Denmark) at 1:60 dilution; and, PMS2, clone M0R4G (Novocastra/Leica Microsystems) at 1:50 dilution. All tests were performed using a Bond Max autostainer (Leica Microsystems) with diaminobenzidine as chromogen for protein-antibody complex visualization. Stains were evaluated by two pathologists (G.R. and E.V.) for all tumour and normal cores, along with external controls for assessing method performance. Each core was evaluated for nuclear staining intensity and distribution of positive cells at 200X and 400X magnification. Cases were considered as pMMR (proficient MMR) if any degree of nuclear expression was observed for all four proteins in the neoplastic cells and dMMR if no IHC nuclear expression was seen for any of the four proteins.
7
Immunohistochemical Analysis of Gastric Cancer
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Tissue specimens from clinical GC samples and xenograft tumors derived from GC cells were fixed with 10% neutralbuffered formalin, and 4-μm paraffin sections were prepared. After rehydration, sections were stained with hematoxylin and eosin for histologic assessment, or were immunostained after antigen retrieval using a Bond-max automated immunostainer (Leica Microsystems, Newcastle upon Tyne, UK). The primary antibodies used were against FOXO1 (1:40, C29H4, Cell Signaling Technology), phospho-FOXO1Ser256 (pFOXO1; 1:60, Cell Signaling Technology), CD31 (1:100, M20, Santa Cruz Biotechnology), HIF-1α (1:50, provided by Dr. Jong-Wan Park at Seoul National University), VEGF (1:200, C1, Santa Cruz Biotechnology), and SIRT1 (1:100, H300, Santa Cruz Biotechnology). Antibody binding was detected with the Bond Polymer Refine Detection Kit (Leica Microsystems). All immunostained sections were lightly counterstained with Mayer’s hematoxylin. Throughout the above analysis, negative controls were prepared by omitting the primary antibody. The results of immunostaining were evaluated by two pathologists (Y.K. and J.-S.P.), who were blinded to the origin of the samples. For statistical analysis, the results of immunostaining for proteins were considered positive if immunoreactivity was seen in ≥ 10% (cytoplasmic pFOXO1 and nuclear SIRT1) or ≥ 5% (nuclear HIF-1α) of the neoplastic cells.
8
Immunohistochemical Analysis of TRKB and BDNF
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Immunohistochemistry (IHC) on human OSCC specimens and mouse tumor tissues was performed using a BOND-MAX autoimmunostainer (Leica Microsystems, Wetzlar, Germany). Deparaffinized and rehydrated sections, which contained the deepest site for each tumor, were subjected to endogenous peroxidase blocking. After heating in an antigen unmasking solution, slides were incubated with the following antibodies: TRKB (1:100; sc-8316, Santa Cruz Biotechnology, CA, USA) and BDNF (1:100; LS-B6557, LifeSpan BioSciences, WA, USA). Color development was carried out using diaminobenzidine tetrahydrochloride, and slides were counterstained with hematoxylin. All samples were stained under the same conditions. For negative controls, primary antibodies were omitted. Human brain histological specimens were used as TRKB and BDNF positive controls. For image analysis, these immunostained sections were scanned using a microscope (BZ-X710, Keyence, Osaka, Japan).
9
Immunohistochemical Profiling of ccRCC Biomarkers
10
Automated Immunostaining and Quantification
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At least three mice from all nine genotypes and ages were taken into analysis. Mice were decapitated with a guillotine and the brains isolated. Brain hemispheres were post-fixed for at least 24 hours in buffered, 4% PFA. Paraffin- embedded, 4-μm-thick coronal sections were stained using a BondMax™ (Leica Microsystems GmbH/Menarini, Germany) automated immunostaining system. Analysis was conducted on 5–10 sections per mouse. Sections were pretreated with Citrate, EDTA or Enzyme 1 pretreatment solutions (Menarini, Germany) and immunostained using anti-IBA1 (EDTA pretreatment 20 min, 1:1,000 for 15 min, Wako GmbH, Germany), anti-GFAP (Enzyme 1 pretreatment, 1:500 for 15 min, DAKO, Germany), anti-NeuN (clone A60, Citrate pretreatment 20min, 1:500 for 15 min, Chemicon, Germany), anti-Ubiquitin (clone Ubi-1, EDTA pretreatment 20 min, 1:10,000 for 15 min, Millipore, Germany) and the Bond™ Polymer Refine Detection kit (Menarini, Germany) as described in (Scheffler et al., 2012). Whole tissue sections were fully digitized at a resolution of 230nm using a Mirax Midi slide scanner (Zeiss, Germany) as described in (Krohn et al., 2011) and 10 fields of view (FOV) at a natural magnification (1:1, 230nm per pixel, 53,3 fold on a 24” screen) were analyzed semi-automatically using the BX Analysis software package and a custom programmed macro (Keyence, Germany).
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