Opal 480

Manufactured by Akoya Biosciences

The Opal-480 is a fluorescence multiplexing platform designed for high-plex immunohistochemistry (IHC) and immunofluorescence (IF) analysis. It enables the simultaneous detection and quantification of up to 7 protein targets in a single tissue section.

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Lab products found in correlation

Opal 570, by Akoya Biosciences (1 mentions) Spectral dapi, by Akoya Biosciences (1 mentions) Opal 520, by Akoya Biosciences (1 mentions) Opal 620, by Akoya Biosciences (1 mentions) Opal 690, by Akoya Biosciences (1 mentions) Cd4 4b12, by Leica (1 mentions) Cd8 c8 144b, by Agilent Technologies (1 mentions) Inform advanced image analysis software inform 2.4.1, by Akoya Biosciences (1 mentions) Cd20 l26, by Roche (1 mentions)

2 protocols using opal 480

Multiplex IHC Protocol for Immune Cells

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Unstained IHC slides from standard IHC preparations were dewaxed (xylene 3 washes × 10 min), gradually rehydrated in EtOH solutions (100% 5 min, 95% 5 min, 70% 2 min), boiled in AR buffer in a microwave, then cooled to RT. Tissues were blocked in PerkinElmer antibody diluent buffer in a humidified chamber at RT for 10 min before overnight staining with primary antibodies, then stained 30 min with HRP-labelled polymeric anti-rabbit antibody. Signal amplification was achieved using Opal™ fluorophore working solutions (1: 100 dilution) at RT for 10 min then microwaved. The primary-secondary-HRP complex was stripped before repeating the steps for multiplexing staining, then mounting the slides with DAPI for 5 min. Slides were visualized using PhenoCharts v1.0.2 (Akoya Bioscience). The antibody diluent (#ARD100), amplification diluent (FP1498), Opal-520-FITC (#FP148700, 1: 100 v/v for perforin), Opal-570-TRITC (#FP148800,1: 200 v/v for NKp46, Opal-480 (#14-0808, 1:200 v/v for CD8) AR6 and AR9 buffer (AR600/900), and Opal-DAPI (FP1490) were purchased form Akoya Bioscience.

Mei K.C., Liao Y.P., Jiang J., Chiang M., Khazaieli M., Liu X., Wang X., Liu Q., Chang C.H., Zhang X., Li J., Ji Y., Melano B., Telesca D., Xia T., Meng H, & Nel A.E. (2020). Liposomal Delivery of Mitoxantrone and a Cholesteryl Indoximod Prodrug Provides Effective Chemo-Immunotherapy in Multiple Solid Tumors. ACS nano, 14(10), 13343-13366.

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Multiplex Immunofluorescence Analysis of TLS

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Multiplexed immunohistofluorescence was performed on TLS-positive cases (identified with HES and CD3/CD20 stainings) using the following antibodies CD20 (L26, Ventana), CD23 (SP23, Ventana), CD21 (2G9, Ventana), CD4 (4B12, Novocastra), CD8 (C8/144B, Dako). Bound primary antibodies were detected using OmniMap anti-Rb HRP (760-4311, Ventana) and OmniMap anti-Ms HRP (760-4310, Ventana) detection kits followed by TSA opal fluorophores (Opal 480, Opal 520, Opal 570, Opal 620 and Opal 690, Akoya Bioscience). The slides were counterstained with spectral DAPI (Akoya Bioscience) and cover-slipped. The slides were scanned using the PerkinElmer Vectra Polaris System, and the multispectral images obtained were unmixed using spectral libraries that were previously built from images stained for each fluorophore (monoplex), using the inForm Advanced Image Analysis software (inForm 2.4.1, Akoya Bioscience) combined with Opal detection kit (Akoya Bioscience).

Vanhersecke L., Brunet M., Guégan J.P., Rey C., Bougouin A., Cousin S., Moulec S.L., Besse B., Loriot Y., Larroquette M., Soubeyran I., Toulmonde M., Roubaud G., Pernot S., Cabart M., Chomy F., Lefevre C., Bourcier K., Kind M., Giglioli I., Sautès-Fridman C., Velasco V., Courgeon F., Oflazoglu E., Savina A., Marabelle A., Soria J.C., Bellera C., Sofeu C., Bessede A., Fridman W.H., Loarer F.L, & Italiano A. (2021). Mature tertiary lymphoid structures predict immune checkpoint inhibitor efficacy in solid tumors independently of PD-L1 expression. Nature cancer, 2(8), 794-802.

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