Bond polymer refine red detection kit

Manufactured by Leica camera

Sourced in Germany

The Bond Polymer Refine Red Detection kit is a specialized laboratory reagent designed for the visualization of target proteins in immunohistochemical and immunocytochemical analyses. The kit utilizes a polymer-based detection system to amplify the signal and enhance the sensitivity of the assay. This product is intended for research use only and its specific applications should be determined by the user.

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15 protocols using bond polymer refine red detection kit

Immunohistochemistry for Soluble Adenylyl Cyclase

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Skin biopsy sections were fixed in 10% NB formalin solution (Avantik, # RS4061) and paraffin embedded. Sections were placed on positively charged slides, and then baked in an oven at 65-70°C for 60 minutes before being loaded onto Leica Bond III, an automated stainer (Leica Microsystems) with Bond Polymer Refine RED Detection Kit (Leica, # DS9390). The sections were pre-treated using heat mediated antigen retrieval with low pH Bond Epitope Retrieval Solution, ER1 (Leica, # AR9961) for 20 minutes. The sections were then incubated for 15 minutes with R21 (Anti-sAC antibody, CEP Biotech, Inc, 1:2500 dilution). Bond Polymer Refine RED Detection Kit (Leica, # DS9390) was used as a chromogen and then counterstained with hematoxylin. Slides were dehydrated and mounted. Slides were imaged on a Nikon light microscope with 10x objective and DS-Fi-1 camera. R21 staining of lymphocytes was scored manually by NSV and GD. For CD3 staining, slides were stained sequentially for R21 as above followed by Leica Bond brown Anti-CD3 stain (Leica, #CD3-565-L-CE) and then hematoxylin.

You J., Reilly M.D., Eljalby M., Bareja R., Yusupova M., Vyas N.S., Bang J., Ding W., Desman G., Miller L.S., Elemento O., Granstein R.D, & Zippin J.H. (2023). Soluble adenylyl cyclase contributes to imiquimod-mediated inflammation and is a potential therapeutic target in psoriasis. Experimental dermatology, 32(7).

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Quantifying Astrocytes and Microglia in CNS

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On day 14 of the study, mice were euthanized and the resulting brains were fixed in 4% paraformaldehyde. Brains were then sliced and processed for immunohistochemistry for glial fibrillar acidic protein (GFAP; astrocyte marker) and ionized calcium-binding adaptor molecule 1 (IBA-1; microglia marker). GFAP was detected using Abcam anti-GFAP (EPR1034Y; 1:200; Abcam, Cambridge, MA) that was visualized by reacting a horseradish peroxidase–conjugated secondary polymer reacted with diaminobenzidine. IBA-1 was detected using Abcam anti-iba-1 (EPR16588; 1:15,000) that was visualized with the Bond Polymer Refine Red Detection Kit (Leica DS9390; Leica Biosystems, Wetzlar, Germany). A hematoxylin counterstain was used to visualize nuclei. Percent area analysis (Visiopharm, Hoersholm, Denmark) was then used to quantitate the extent of GFAP and IBA-1 expression in the hippocampus and cerebral cortex (three slices per animal; N = 5 for each group).

Dickerman R.D., Williamson J., Mathew E., Butt C.M., Bird C.W., Hood L.E, & Grimshaw V. (2022). Branched-Chain Amino Acids Are Neuroprotective Against Traumatic Brain Injury and Enhance Rate of Recovery: Prophylactic Role for Contact Sports and Emergent Use. Neurotrauma Reports, 3(1), 321-332.

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Dual Immunostaining of α-Synuclein and Tau

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Briefly, a sequential dual immunostaining protocol was used for the simultaneous visualization of α-synuclein and tau. The colorimetric detection of α-synuclein was carried out firstly using a Bond Polymer Refine DAB detection kit (Leica) resulting in a brown precipitate. Next, the Bond Polymer Refine Red detection kit (Leica) was used to visualize the expression of tau protein by means of a red-colored precipitate.

Erro Aguirre M.E., Zelaya M.V., Sánchez Ruiz de Gordoa J., Tuñón M.T, & Lanciego J.L. (2015). Midbrain catecholaminergic neurons co-express α-synuclein and tau in progressive supranuclear palsy. Frontiers in Neuroanatomy, 9, 25.

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Immunohistochemistry of Lung Tissues

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FFPE mouse and/or human lung tissues were stained with antibodies against CD79α-B cell marker (M7050, Dako), B-cell activating factor-BAFF (Ab117256, Abcam) and CD21-follicular dendritic cell marker (Ab75985, Abcam) and Ki67-proliferation marker (Ab16667, Abcam) using the Bond Polymer Refine Red Detection kit on the Leica Bond Autostainer according to the manufacturer’s protocol as previously described [16 (link)]. Slides were scanned using the Aperio imaging system (Leica Biosystem; Concord, Ontario).

Tam A., Tanabe N., Churg A., Wright J.L., Hogg J.C, & Sin D.D. (2020). Sex differences in lymphoid follicles in COPD airways. Respiratory Research, 21, 46.

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PDL-1 Immunohistochemistry in Tumor Tissue

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Tumors were harvested and flash-frozen in liquid nitrogen. Frozen tissue sections were cut at 5 μm onto coated slides. Sections were air-dried overnight and then fixed in 10% neutral buffered formalin (NBF) for 5 min before being treated with 1% H₂O₂ in dH₂O for 15 min at room temperature. Slides were then washed in dH₂O to remove excess H₂O₂. Slides were rinsed in Bond Wash (Leica) and placed on the Leica Bond Automated stainer. The slides were stained with Rat primary PDL-1 (13-5982-52; EBio) 1:500 in Animal Free Diluent (SP-5035; Vector Labs). The BOND Polymer Refine Red Detection kit (Leica) was used according to the manufacturer’s protocol. The slides were then digitized according to a previously described protocol.⁵⁷ (link)

El-Sayes N., Walsh S., Vito A., Reihani A., Ask K., Wan Y, & Mossman K. (2022). IFNAR blockade synergizes with oncolytic VSV to prevent virus-mediated PD-L1 expression and promote antitumor T cell activity. Molecular Therapy Oncolytics, 25, 16-30.

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Immunohistochemical Analysis of ATM in FFPE UM

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Sections (4 µm thick) from FFPE UM were deparaffinized and underwent heat induced epitope retrieval at 96 °C, pH 9.0 for 20 min on the Leica Bond RXm, followed by staining using the Leica Bond polymer refine red detection kit, according to the standard manufacturer’s protocols. A mouse monoclonal anti-ATM antibody (ab78; Abcam, Cambridge, UK) was used at 1:100 dilution. Sections were counter-stained with hematoxylin, dehydrated and mounted using a solvent based mountant. Negative controls used mouse IgG1 at the same concentration as the primary antibody. Staining was assessed and scored by three individuals (R.N.H., S.E.C., and J.K.). Statistical analysis was performed using a one sample t-test.

Hussain R.N., Coupland S.E., Khzouz J., Kalirai H, & Parsons J.L. (2020). Inhibition of ATM Increases the Radiosensitivity of Uveal Melanoma Cells to Photons and Protons. Cancers, 12(6), 1388.

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Quantifying Immune Cell Infiltration in Melanoma

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Single-marker IHC was performed on formalin-fixed paraffin-embedded (FFPE) tumor sample sections, (4 μm thickness) after heat-induced antigen retrieval (HIER, citrate buffer, pH 6.0), obtained from patients with melanoma at the designated treatment timepoints. Granzyme B was localized by immuno-labeling tissue sections with mouse mAb (clone GrB-7; DAKO) at 1:25 dilution followed by detection with the EnVisionTM G2 System/AP kit and visualization by Permanent Red Chromogen substrate staining. CD8 was labeled in FFPE tissue sections using mouse monoclonal anti-CD8 antibody (clone C8/144B; DAKO) at a 1:100 dilution followed by detection with the BOND Polymer Refine Red Detection Kit (Leica) and AP Red chromogen substrate visualization. Whole-slide scans were imaged using the Aperio-AT system (Leica Biosystems) and transferred into the HALO (Indica Labs) platform for quantitative digital image analysis.

Andtbacka R.H., Wang Y., Pierce R.H., Campbell J.S., Yushak M., Milhem M., Ross M., Niland K., Arbeit R.D., Parasuraman S., Bickley K., Yeung C.C., Aicher L.D., Smythe K.S, & Gan L. (2022). Mavorixafor, an Orally Bioavailable CXCR4 Antagonist, Increases Immune Cell Infiltration and Inflammatory Status of Tumor Microenvironment in Patients with Melanoma. Cancer Research Communications, 2(8), 904-913.

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Immunohistochemical Analysis of Duodenal Tissues

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Duodenal segments were fixed in 10% buffered formalin and embedded in paraffin (FFPE) prior to sectioning and staining with H&E. 5 um FFPE sections were deparaffinized, rehydrated, and placed on a Leica Bond Rx immunohistochemical stainer for heat-activated antigen retrieval and sequential staining with Rabbit anti-human CD3 (Lab Vision), the Bond Polymer Refine Detection kit (Leica), and Deep Space Black Chromogen (Biocare Medical). Slides were then incubated with rabbit anti-IBA1 (Wako) and the Bond Polymer Refine Red Detection kit (Leica) and were counterstained with methyl-green. Isotype matched antibodies were used on serial tissue sections as negative controls.

Paustian A.M., Paez-Cortez J., Bryant S., Westmoreland S., Waegell W, & Kingsbury G. (2017). Continuous IL-23 stimulation drives ILC3 depletion in the upper GI tract and, in combination with TNFα, induces robust activation and a phenotypic switch of ILC3. PLoS ONE, 12(8), e0182841.

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Quantifying Airway Epithelial Markers in Mouse Lungs

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Formalin-fixed paraffin-embedded (FFPE) mouse lung tissues were stained with antibodies against Ptch1 (06-1102; EMD Millipore) and Ki67 (ab16667; Abcam) using the Bond Polymer Refine Red Detection kit on the Leica Bond Autostainer according to the manufacturer’s protocol. Formalin-fixed paraffin-embedded (FFPE) mouse lung tissues were stained with antibody against MUC5AC (MA1-21907, ThermoFisher) using the high sensitivity universal detection system-MACH kit (Biocare Medical, Concord, CA) as previously described^{50 (link)}.
Total airway epithelial mucous expression was evaluated by staining FFPE-mouse lung tissues with PAS staining. Total epithelial-specific PAS intensity and epithelial thickness were normalized to the length of the BM. Airway epithelial-specific Ki67+ positive cells and Ptch1 protein intensity were normalized to the length of the BM. All slides were scanned using the Aperio imaging system and color intensities were quantified using the Aperio ScanScope software.

Tam A., Hughes M., McNagny K.M., Obeidat M., Hackett T.L., Leung J.M., Shaipanich T., Dorscheid D.R., Singhera G.K., Yang C.W., Paré P.D., Hogg J.C., Nickle D, & Sin D.D. (2019). Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease. Scientific Reports, 9, 3353.

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Quantifying PTCH1 and Ki67 in FFPE Human Lung Tissues

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Formalin-fixed paraffin-embedded (FFPE) human lung tissues were stained with antibodies against PTCH1 (06-1102; EMD Millipore) and Ki67 (ab16667; Abcam) using the Bond Polymer Refine Red Detection kit on the Leica Bond Autostainer according to the manufacturer’s protocol. Slides were scanned using the Aperio imaging system (Leica Biosystem; Concord, Ontario). Airway epithelial-specific PTCH1 protein intensity was quantified using the Aperio imaging system, while the Ki67+ cells were manually counted and normalized to the length of the basement membrane.

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