Bond 3 autostainer

Manufactured by Leica

Sourced in United States, Germany, United Kingdom

The Bond III Autostainer is a fully automated platform designed for immunohistochemistry (IHC) and in situ hybridization (ISH) staining. It provides a standardized and consistent staining process for tissue samples. The Bond III Autostainer automates the entire staining workflow, including dewaxing, epitope retrieval, primary antibody incubation, and detection, ensuring reliable and reproducible results.

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Lab products found in correlation

Bond polymer refine detection kit, by Leica (12 mentions) Cytoseal xyl, by Thermo Fisher Scientific (4 mentions) Bond epitope retrieval solution 2, by Leica (4 mentions) Bond polymer refine detection, by Leica (3 mentions) Anti pgp9.5 rabbit polyclonal, by Agilent Technologies (2 mentions) Mib 1, by Agilent Technologies (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) Aperio at2 scanner, by Leica (2 mentions) Bond polymer refine red detection kit, by Leica (2 mentions) Bond ready to use ish eber probe, by Leica (2 mentions) Link48 autostainer, by Agilent Technologies (2 mentions) Epr17341, by Abcam (1 mentions) Clone mum1p, by Agilent Technologies (1 mentions) Clone cs 1 4, by Agilent Technologies (1 mentions) Biotinylated link universal, by Agilent Technologies (1 mentions) Ab124824, by Abcam (1 mentions) Eclipse e400 microscope, by Nikon (1 mentions) Axio microscope, by Zeiss (1 mentions) Eos 600d digital camera, by Canon (1 mentions) Clone l26, by Agilent Technologies (1 mentions)

60 protocols using bond 3 autostainer

Immunohistochemical Profiling of Endometrial Ovarian Cancer

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ER and PR expression data were available for 90 of the 112 EnOC cases characterised by WES (Fig. 1)^{24 (link)}. IHC for PR and ER was performed on human tissue microarrays comprising three 0.8 mm cores per case, as previously described^{24 (link)}. IHC used 1:50 mouse anti-PR antibody M3569 clone PgR-636 and 1:50 rabbit anti-ER antibody M3643 clone EP1; PR and ER IHC was performed on the Leica BOND III Autostainer and expression was assessed by histoscore, a quantitative nuclear expression score incorporating staining intensity and proportion of positive tumour nuclei^{31 (link)}. HREP-based subtype was available for 74 cases from the unsupervised subtyping study^{24 (link)};16 further cases were classified based on their PR and ER histoscore using a threshold of histoscore ≥150 for positivity, as indicated by the unsupervised subtyping study^{24 (link)} (Fig. 1).
p53 IHC data were available for 87 of the 90 cases^{23 (link)}. IHC was performed on the Leica BOND III Autostainer using a 1:50 dilution of p53 antibody (clone DO-7, DAKO). Wild-type pattern was defined as variable nuclear staining intensity, while aberrant staining was defined as strong diffuse nuclear positivity (aberrant positive) or complete absence of nuclear staining (aberrant null).

Hollis R.L., Stanley B., Thomson J.P., Churchman M., Croy I., Rye T., Bartos C., Nussey F., Mackean M., Meynert A.M., Semple C.A., Gourley C, & Herrington C.S. (2021). Integrated molecular characterisation of endometrioid ovarian carcinoma identifies opportunities for stratification. NPJ Precision Oncology, 5, 47.

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Immunohistochemical Detection of Pan-TRK

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Staining was performed on a BOND III autostainer (Leica Instruments, Wetzlar, Germany). Antigen retrieval at pH 9.0 was performed for 60 min. The primary pan-TRK antibody, EPR17341 (Abcam, Cambridge, UK) was diluted 1:100 in DAKO background-reducing diluent and incubated at 22 °C for 30 min. Detection was performed using a Bond Polymer Refine Detection kit and 3,3'-diaminobenzidine chromagen. On slide controls for IHC were appendix (cytoplasmic, membranous, and nuclear in peripheral nerves and ganglia, negative staining in epithelia) and cerebellum (membranous and cytoplasmic staining in neurons).

Silvertown J.D., Lisle C., Semenuk L., Knapp C., Jaynes J., Berg D., Kaul N., Lachapelle J., Richardson L., Speevak M., Sarras H., Berman D.M., Carter R., Feilotter H, & Feltis T. (2022). Prevalence of NTRK Fusions in Canadian Solid Tumour Cancer Patients. Molecular Diagnosis & Therapy, 27(1), 87-103.

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CMV Infection Detection and Management

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The primary outcome was reoperation after the primary operation, and the secondary outcome was in-hospital mortality due to CMV infection. CMV infection was diagnosed using immunohistochemical staining of the surgical specimen and evaluated by experienced pathologists. The results of enrolled patients were reviewed by an expert pathology specialist. Immunohistochemical stains for CMV were performed on 2-µm tissue sections. Deparaffinized slides were stained with anti-CMV (mouse monoclonal antibody, clones CCH2 and DDG9, 1:40 dilution; Dako). Slides were incubated in the Bond Epitope Retrieval Solution 2 at 100 ℃ for 20 minutes and stained on the Bond III Autostainer (Leica Microsystems). CMV infection in the blood was detected using a CMV pp65 antigenemia assay with immunofluorescence staining. CMV infection was defined as a CMV pp65 antigen-positive cell number greater than 10 positive cells per 400,000 WBCs. In cases wherein CMV infection was detected in the surgical specimen, antiviral induction therapy was initiated with intravenous ganciclovir (5 mg/kg, twice daily for 2 weeks, with renal dose adjustment if needed) [18 (link)]. If the patient was discharged before the expected treatment date, oral valganciclovir was prescribed (1 g, 3 times daily). The treatment period was extended depending on the patient’s condition.

Kim S., Yoon K.W., Gil E., Yoo K., Choi K.J, & Park C.M. (2023). Emergency gastrointestinal tract operation associated with cytomegalovirus infection. Annals of Surgical Treatment and Research, 104(2), 119-125.

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Paraffin-Embedded Tissue Immunohistochemistry

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Chromogenic immunohistochemistry (IHC) was performed on paraffin-embedded tissues that were sectioned at 5 micrometers. All IHC was carried out in the Bond III Autostainer (Leica Microsystems Inc.). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Antigen retrieval was performed for 20 min at 100ºC in Bond-Epitope Retrieval solution 1, pH-6.0 (AR9961).

Murphy R.M., Tasoulas J., Porrello A., Carper M.B., Tsai Y.H., Coffey A.R., Kumar S., Zeng P.Y., Schrank T.P., Midkiff B.R., Cohen S., Salazar A.H., Hayward M.C., Hayes D.N., Olshan A., Gupta G.P., Nichols A.C., Yarbrough W.G., Pecot C.V, & Amelio A.L. (2022). Tumor Cell Extrinsic Synaptogyrin 3 Expression as a Diagnostic and Prognostic Biomarker in Head and Neck Cancer. Cancer Research Communications, 2(9), 987-1004.

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Histopathological Analysis of Cutaneous KLIP

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Formalin-fixed, paraffin-embedded skin biopsies were studied. Three-micrometer-thick sections were stained with hematoxylin–eosin–saffron (HES) and pH 2.5 Alcian blue stain, and immunohistochemical analyses used monoclonal antibodies to CD3, CD4, CD8, CD20, CD68, CD123, granzyme B, and myeloperoxidase (MPO) (Dako, France). We used a standard avidin–biotin–peroxidase method with diaminobenzidine (DAB) chromogen, and the BOND-III Autostainer (Leica Microsystems, Newcastle-upon, Tyne, UK), after antigen retrieval by heating in the appropriate buffer. All skin biopsies were examined and interpreted by the same pathologist (NO).
KLIP was defined as a dermal infiltrate, or foci within dermal infiltrates, composed of mononuclear cells and nuclear debris, without neutrophils. Immunohistochemically labeled mononuclear cells were CD163⁺ macrophages, some of which were MPO⁺ cells, CD8⁺ lymphocytes, including cytotoxic (granzyme B+) cells, and CD123⁺ PDC (Fig. 1A–D).

Thai L.H., Ingen-Housz-Oro S., Godeau B., Rethers L., Wolkenstein P., Limal N., Papillon V., Kapfer J., Chosidow O, & Ortonne N. (2015). Kikuchi Disease-Like Inflammatory Pattern in Cutaneous Inflammatory Infiltrates Without Lymph Node Involvement: A New Clue for the Diagnosis of Lupus?. Medicine, 94(46), e2065.

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Evaluating MMP14 Expression in NSCLC

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For evaluation of MMP14 expression in NSCLC patients we used TMAs derived from 210 lung cancer patients from WCMC. Seventy-four percent of the patients were at stage IA/IB, 8% at stages IIA/IIB, 12% at stage III and 6% at stage IV. Immunohistochemical staining of MMP14 (Clone LEM-2/63.1, Abcam) was performed using the Bond III Autostainer (Leica Microsystems, IL, USA). TMAs were examined in a double blinded manner by two individuals using scale 0 to 3 with score >1 considered positive.

Stawowczyk M., Wellenstein M.D., Lee S.B., Yomtoubian S., Durrans A., Choi H., Narula N., Altorki N.K., Gao D, & Mittal V. (2016). Matrix Metalloproteinase 14 promotes lung cancer by cleavage of Heparin-Binding EGF-like Growth Factor. Neoplasia (New York, N.Y.), 19(2), 55-64.

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Paraffin-Embedded Tissue IHC on Bond III

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Chromogenic IHC was performed on paraffin-embedded tissues that were sectioned at 5 μm. All IHC was carried out in the Bond III Autostainer (Leica Microsystems Inc.). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Antigen retrieval was performed for 20 minutes at 100°C in Bond-Epitope Retrieval solution 1, pH-6.0 (AR9961).

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Immunohistochemical Characterization of B-cell Markers

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Immunohistochemical staining of LMP1 (1:50, clone CS.1–4; Dako, Carpinteria, CA, USA), EBNA2 (1:50, clone PE2; Dako), BCL6 (1:10, clone PG-B6p; Dako), PRDM1 (1:50, clone 3H2E8; Santa Cruz Biotechnology, Dallas, TX, USA) and MUM1/IRF4 (1:100, clone MUM1p; Dako) were accomplished using the Bond III Autostainer (Leica Microsystems, Buffalo Grove, IL, USA). Formalin-fixed, paraffin-embedded tissues or cell block sections were first baked and deparaffinized. Antigens were then retrieved by heating the slides at 37 °C in Bond Enzyme solution (Leica Microsystems) for 10 min (for LMP1), and at 99–100 °C in Bond Epitope Retrieval Solution 2 for 20 (for EBNA2, PRDM1 and MUM1) or 30 min (for BCL6). Sections were then incubated sequentially with the endogenous peroxidase block, primary antibody, postprimary (equivalent to secondary antibody), polymer (equivalent to tertiary antibody), diaminobenzidine and hematoxylin for 5, 25, 15, 25, 10 and 5 min, respectively. Bond Polymer Define Detection (Leica Microsystems) was used for EBNA2 and MUM1, and Bond Polymer Refine Detection (Leica Microsystems) was used for LMP1, BCL6 and PRDM1. Finally, the stained slides were dehydrated and mounted in Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI, USA).

Zhang T., Ma J., Nie K., Yan J., Liu Y., Bacchi C.E., Queiroga E.M., Gualco G., Sample J.T., Orazi A., Knowles D.M, & Tam W. (2014). Hypermethylation of the tumor suppressor gene PRDM1/Blimp-1 supports a pathogenetic role in EBV-positive Burkitt lymphoma. Blood Cancer Journal, 4(11), e261-.

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Immunohistochemical Detection of Axl Receptor

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Immunohistochemical staining of Axl (goat polyclonal, dilution 1:40, R&D Systems) was accomplished using the Bond III Autostainer (Leica Microsystems, Illinois, USA). Formalin-fixed, paraffin-embedded tissue sections were first baked and deparaffinised. Antigen retrieval was accomplished by heating the slides at 99–100°C in Bond Epitope Retrieval Solution 1 for 30 min. Sections were then incubated sequentially with endogenous peroxidase block for 5 min, primary antibody for 30 min, Biotinylated Link Universal (Dako) for 25 min, Streptavidin-HRP for 25 min, diaminobenzidine (DAB) for 10 min, and haematoxylin for 5 min. Finally, the sections were dehydrated in 100% ethanol, and mounted in Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, Michigan, USA). Normal breast tissue, which was used as a positive control, shows membranous Axl staining of luminal ductal cells with variable cytoplasmic staining.

D’Alfonso T.M., Hannah J., Chen Z., Liu Y., Zhou P, & Shin S.J. (2014). Axl receptor tyrosine kinase expression in breast cancer. Journal of clinical pathology, 67(8), 690-696.

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CXCR4 Expression in FFPE Tissue Samples

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Three-micron-thick formalin-fixed paraffin-embedded (FFPE) tissue sections of normal human tonsil, placenta and neuroendocrine tumours were subjected to conventional single immunohistochemistry (IHC) to study the expression of CXCR4 using a rabbit monoclonal anti-CXCR4 antibody [UMB2, Abcam, Ab124824]. Human tonsil and placenta tissues were used as the positive control tissue to define the optimal staining protocol, and a dilution of 1:50 was found to result in a specific staining pattern in the absence of a background signal. Parallel tests without the primary antibody served as the negative control. Immunostaining was performed using the automated BOND-III Autostainer (Leica Microsystems, UK) according to protocols described elsewhere.^{21 (link)} The semi-quantitative analysis of the stained sections was performed by an expert pathologist having no knowledge of the pathological data using a Nikon Eclipse E400 microscope. Immunoreactivity was quantified in terms of the percentage of positive tumour cells and staining intensity.

Rizzo F.M., Vesely C., Childs A., Marafioti T., Khan M.S., Mandair D., Cives M., Ensell L., Lowe H., Akarca A.U., Luong T., Caplin M., Toumpanakis C., Krell D., Thirlwell C., Silvestris F., Hartley J.A, & Meyer T. (2019). Circulating tumour cells and their association with bone metastases in patients with neuroendocrine tumours. British Journal of Cancer, 120(3), 294-300.

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