Opal 7 color kit

Manufactured by Akoya Biosciences

Sourced in United States

The Opal 7-Color Kit is an immunohistochemistry reagent designed for multiplexed analysis of up to 7 protein targets within a single tissue section. The kit includes the necessary components to perform the staining protocol, such as fluorophore-conjugated antibodies and ancillary reagents. The core function of the Opal 7-Color Kit is to enable the simultaneous visualization and quantification of multiple biomarkers in a single sample.

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Lab products found in correlation

Vectra multispectral microscope, by Akoya Biosciences (1 mentions) Vectra multispectral imaging system, by Akoya Biosciences (1 mentions) Inform 2.4.8 image analysis software, by Akoya Biosciences (1 mentions) Target retrieval solution, by Agilent Technologies (1 mentions) Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (1 mentions) Opal fluorophore conjugated tyramide signal amplification reagent, by Akoya Biosciences (1 mentions) Ab7753, by Abcam (1 mentions) Inform software, by PerkinElmer (1 mentions) Dapi d9542, by Merck Group (1 mentions) Mantra system, by PerkinElmer (1 mentions) Pannoramic midi, by 3DHISTECH (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)

5 protocols using opal 7 color kit

Multiplexed Immunofluorescence Staining for FFPE Tissue Microarray

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Development and optimization of the mIF platform has been previously described (Parra et al., 2020 ). mIF staining was performed in a 4-μm-thick section obtained from a FFPE TMA block, using the Opal 7-Color Kit (Akoya Biosciences, Waltham, MA, USA) and scanned using a Vectra multispectral microscope (Akoya Biosciences). The IF markers used were pan-Cytokeratin (CK, clone AE1/AE3, (DAKO) and Alpha-SMA ab5694 (Abcam). Multiplexed stained sections were imaged using the VECTRA multispectral imaging system (Akoya Biosciences). TMA cores were annotated using InForm 2.4.8 image analysis software (Akoya Biosciences).

Satpathy S., Krug K., Jean Beltran P.M., Savage S.R., Petralia F., Kumar-Sinha C., Dou Y., Reva B., Kane M.H., Avanessian S.C., Vasaikar S.V., Krek A., Lei J.T., Jaehnig E.J., Omelchenko T., Geffen Y., Bergstrom E.J., Stathias V., Christianson K.E., Heiman D.I., Cieslik M.P., Cao S., Song X., Ji J., Liu W., Li K., Wen B., Li Y., Gümüş Z.H., Selvan M.E., Soundararajan R., Visal T.H., Raso M.G., Parra E.R., Babur Ö., Vats P., Anand S., Schraink T., Cornwell M., Rodrigues F.M., Zhu H., Mo C.K., Zhang Y., da Veiga Leprevost F., Huang C., Chinnaiyan A.M., Wyczalkowski M.A., Omenn G.S., Newton C.J., Schurer S., Ruggles K.V., Fenyö D., Jewell S.D., Thiagarajan M., Mesri M., Rodriguez H., Mani S.A., Udeshi N.D., Getz G., Suh J., Li Q.K., Hostetter G., Paik P.K., Dhanasekaran S.M., Govindan R., Ding L., Robles A.I., Clauser K.R., Nesvizhskii A.I., Wang P., Carr S.A., Zhang B., Mani D.R, & Gillette M.A. (2021). A proteogenomic portrait of lung squamous cell carcinoma. Cell, 184(16), 4348-4371.e40.

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Multiplex Immunostaining of Mouse Lung

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Paraformaldehyde-fixed, paraffin-embedded mouse lungs were sectioned. After deparaffinization and rehydration, antigens were retrieved by boiling in Dako target retrieval solution (#S1699) for 15 min. Endogenous peroxidase was blocked by incubating in 3% hydrogen peroxide for 30 min, and sections were then blocked by 5% BSA in Tris-buffered saline + polysorbate 20 (TBST) for 1 h at room temperature. For IHC staining, sections were incubated with primary antibodies diluted in 2.5% BSA in TBST at 4 °C overnight, then with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Finally, results were visualized by using a Dako liquid DAB+ substrate chromogen system (K3468). For CD3⁺, CD8⁺, and RelB multiplex immunofluorescence staining, an Opal 7-color kit (Akoya Biosciences, #NEL811001KT) was used according to the manufacturer’s protocol. See Supplementary Data 4 for antibody information.

Zhao M., Wang T., Gleber-Netto F.O., Chen Z., McGrail D.J., Gomez J.A., Ju W., Gadhikar M.A., Ma W., Shen L., Wang Q., Tang X., Pathak S., Raso M.G., Burks J.K., Lin S.Y., Wang J., Multani A.S., Pickering C.R., Chen J., Myers J.N, & Zhou G. (2024). Mutant p53 gains oncogenic functions through a chromosomal instability-induced cytosolic DNA response. Nature Communications, 15, 180.

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Multiplex Immunofluorescence for Immune Cell Profiling

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Using an Opal 7-color Kit (Akoya Biosciences, Marlborough, MA, USA), multiplex immunofluorescence staining was conducted on TMA sections according to the manufacturer’s protocol and as previously described by Yeong et al. (24 (link)) for simultaneous detection of CD68, myeloperoxidase (MPO), citrullinated histone H3, and DAPI. The TMA sections were baked at 65°C for 2 h and subjected to deparaffinization, rehydration, and heat-induced epitope retrieval. The sections were then incubated with a primary antibody for 1 h at room temperature, followed by incubation with an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Akoya Biosciences). We incubated the slides with an opal fluorophore-conjugated tyramide signal amplification reagent (Akoya Biosciences). Heat-induced epitope retrieval was performed to remove the bound antibody complexes. The same procedure was repeated until all targets were detected, and the samples were labeled with DAPI (Akoya Biosciences). The following antibodies were used: anti-CD68 (D4B9C; Cell Signaling Technology, Danvers, MA, USA)/Opal 520, anti-citrullinated histone H3/Opal 620 (ab5103; Abcam, Cambridge, UK), MPO (E1E7I; Cell Signaling Technology, Danvers, MA, USA)/Opal 690. Finally, the sections were mounted using a hard-set medium.

Chen X., Ma H., Mo S., Yu S., Lu Z, & Chen J. (2022). Intratumoral neutrophil extracellular traps are associated with unfavorable clinical outcomes and immunogenic context in pancreatic ductal adenocarcinoma. Frontiers in Immunology, 13, 1027459.

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Multiplex IHC for M1/M2 and Teffs

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An Opal 7‐color Kit (NEL871001KT, Akoya Bioscience, USA) was used to perform multiplex immunohistochemical staining to identify M1 and M2 macrophages (CD68/CD163/CD206/INOS). Effector T cells (Teffs; GZMB/CD8) were characterized separately in two separate panels using an Opal 4‐color Kit (NEL840001KT, Akoya Bioscience, USA). Labeling of multi‐cytokeratin using panCK (ab7753, Abcam, UK) in all panels was used to segment the tumor nest and stroma or determine the IM. The nuclei of all cells were stained with DAPI (D9542; Sigma‐Aldrich, USA). The details of the primary antibodies are presented in Table S9, Supporting Information.
A Mantra System (PerkinElmer, USA) was used to capture multispectral panoramic images after staining. The scanned slides were then analyzed using the InForm software (PerkinElmer, USA) to obtain quantitative data on the region of interest (ROI). InForm can accurately count positive cells and identify them by setting reasonable thresholds, which allowed for the computation of the density and ratio of the target cells in the ROI. It also allowed automated segmentation of the tumor nest (panCK⁺) and stroma (panCK⁻) to collect quantitative data from different ROIs.

Wang J., Qiu Q., Zheng Q., Zhao Y., Xu Y., Zhang T., Wang S., Wang Q., Jin Q., Ye Y., Li P., Xie J., Lin J., Lu J., Chen Q., Cao L., Yang Y., Zheng C, & Huang C. (2023). Tumor Immunophenotyping‐Derived Signature Identifies Prognosis and Neoadjuvant Immunotherapeutic Responsiveness in Gastric Cancer. Advanced Science, 10(15), 2207417.

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Multiparametric Immunofluorescence Staining

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We performed mIF staining as previously described [12] (link). In brief, the paraffin-embedded slides were heated, deparaffinized using xylene, and rehydrated in graded alcohols. After antigen retrieval and blocking, the primary antibody (Table S1) was applied and incubated at 4℃ overnight. Opal polymer horseradish peroxidase (HRP) was used as the secondary antibody. The slides were washed, and tyramide signal amplification (TSA) dye (Opal 7 Color Kit, Akoya Biosciences) was applied. The slides were then microwaved to strip the primary and secondary antibodies, washed, and blocked again using blocking solution. Afterward, a second primary antibody and 4′,6′-diamidino-2-phenylindole (DAPI) were applied. Finally, slides were coverslipped using ProLong Gold Antifade Reagent (P36930, Invitrogen).
Whole slide scans were performed using a multispectral microscope (Pannoramic MIDI, 3DHISTECH), and were imaged to observe the landscape of ABCB11 and CYP1A1 expression, as well as infiltration of Treg and T helper 2 (Th2) cells. Briefly, ABCB11⁺ cells>10% in a TLS was defined as ABCB11⁺TLS. CYP1A2⁺ cells>10% in a TLS was defined as CYP1A2⁺TLS. Cells with colocalization of ABCB11 and CYP1A2 >10% in a TLS was defined as ABCB11⁺CYP1A2⁺TLS. Treg cells were counted for positively stained FOXP3 cells per 200 × magnification.

Ning J., Hao J., Guo F., Hou X., Li L., Wang J., Wang S., Gao Y., Zheng X, & Gao M. (2023). ABCB11 accumulated in immature tertiary lymphoid structures participates in xenobiotic metabolic process and predicts resistance to PD-1/PD-L1 inhibitors in head and neck squamous cell carcinoma. Translational Oncology, 36, 101747.

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