Opal 650

Manufactured by Akoya Biosciences

Sourced in Japan, United States

The Opal 650 is a fluorescent labeling reagent that can be used to detect and visualize specific proteins or other biomolecules in biological samples.

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Lab products found in correlation

13 protocols using opal 650

Single-Molecule RNA FISH in Mouse Embryos

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smRNA-FISH was performed according to the protocol of the RNAscope Multiplex Fluorescent v2 Assay (ACD-bio. 323110). In brief, embryos were fixed for 26-30h in 4%paraformaldehyde at 4°C and then dehydrated in methanol. For smRNA-FISH on sections at E6.5-E7.5, the decidua was embedded in paraffin after fixation, dehydrated in methanol and incubated for 16h in butanol at 4°C. For smRNA-FISH on sections of E9.5 chimera, the embryos were embedded in histogel (Epredia HG-4000-012) prior to paraffin embedding. Tissue sections were cut at 7 to 10 μm. Whole-mount smRNA-FISH was performed as previously described ^{72 (link)}. Probes are reported in Supplementary Table 5. To develop probes, we have used Opal dyes from Akoya Bioscience (Opal-520, Opal-570 and Opal-650, from 1:100 to 1:200). Embryos or sections were imaged using an AxioZoom.V16 microscope (Zeiss) or an LSM800 confocal microscope (Zeiss). When analysing litters from Mesp1-Cre or Zic3 KO lines, embryos were genotyped by PCR after imaging.

Lin X., Swedlund B., Ton M.L., Ghazanfar S., Guibentif C., Paulissen C., Baudelet E., Plaindoux E., Achouri Y., Calonne E., Dubois C., Mansfield W., Zaffran S., Marioni J.C., Fuks F., Göttgens B., Lescroart F, & Blanpain C. (2022). Mesp1 controls the chromatin and enhancer landscapes essential for spatiotemporal patterning of early cardiovascular progenitors. Nature cell biology, 24(7), 1114-1128.

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Multimodal Spleen and Liver Tissue Analysis

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3-μm-thick spleen and liver sections were produced from formalin-fixed, paraffin-embedded tissue blocks. Consecutive sections were subjected to Giemsa staining according to standard protocols, immunohistochemistry for CD30 and Ki67 using the Ultra-View 3,3′-Diaminobenzidine (DAB) universal staining kit (Roche), and detection of EBER 1 by in situ hybridization (EBER 1 DNP Probe, Ventana/Roche Tissue Diagnostics, Tucson, AZ, USA) on the automated Benchmark Ultra instrument (Ventana/Roche Tissue Diagnostics, as described previously).³² (link) Multiplexed immunohistochemistry was performed using the Opal technology (Akoya Bioscience, Menlo Park, CA) for detection of CD3 (polyclonal; Dako, Bollschweil, Germany), CD20 (clone L26, Dako), and Ki67 (Thermo Scientific, Waltham, MA, USA). The detection reagents Opal 650, Opal 690, and Opal 620 (Akoya Bioscience) were applied (Table S8). Multiplexed immunohistochemistry was analyzed by the VectraPolaris instrument (Akoya Biosciences). For quantitative analysis, multiplexed images were analyzed using inForm 2.4.8 software (Akoya Biosciences) as described previously.³² (link) Several representative regions of interest (ROIs) were selected from the spleen (2–4 ROIs per sample) and liver (3–6 per sample, near blood vessels). ROIs were scanned at 5,568 × 4,176 pixel or 3,728 × 2 729 pixel at 0.5 or 0.25 μm/pixel resolution.

Slabik C., Kalbarczyk M., Danisch S., Zeidler R., Klawonn F., Volk V., Krönke N., Feuerhake F., Ferreira de Figueiredo C., Blasczyk R., Olbrich H., Theobald S.J., Schneider A., Ganser A., von Kaisenberg C., Lienenklaus S., Bleich A., Hammerschmidt W, & Stripecke R. (2020). CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease. Molecular Therapy Oncolytics, 18, 504-524.

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Single-Molecule RNA FISH in Mouse Embryos

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Multiplexed In Situ Analysis of ECM Proteins

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Probes and reagents were purchased from Advanced Cellular Diagnostics (ACD, 323136 and 323110) following the manufacturer’s protocol. Cryosections (16 μm) were prepared per ACD’s protocol. Pepsin (Sigma-Aldrich, R2283) was used for antigen retrieval followed by mRNA probe hybridization (ACD, 439101 [ACAN]; ACD, 407221-C [Col2]; Opal dyes: Akoya Biosciences, Opal 520, FP1487001KT; Opal 650, FP1496001KT) and immunofluorescent staining to recover tdTomato protein digested by Pepsin, using the anti-RFP antibody staining protocol described in Immunofluorescent staining above. The fluorescence assays were designed based on selection of fluorescent dyes with distinct excitation and emission spectra per manufacturer database (Invitrogen, Akoya Biosciences) and https://www.fpbase.org and based on optimization assays to ensure a sufficient signal-to-noise ratio and spectral separation to comply with ACD and Leica confocal imaging guidelines.

Massengale M., Massengale J.L., Benson C.R., Barywano N., Oki T., Steinhauser M.L., Wang A., Balani D., Oh L.S., Randolph M.A., Gill TJ I.I.I., Kronenberg H.M, & Scadden D.T. (2023). Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo. JCI Insight, 8(17), e167858.

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Multiplexed Immunofluorescence Staining of FFPE TMAs

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Multiplexed fluorescence staining was performed as previously described (39 (link) {Song, 2022 #205)}. In brief, 4-μm FFPE TMAs sections were deparaffinized in xylene and then rehydrated in 100, 90, and 70% alcohol successively. Antigen unmasking was performed with a preheated epitope retrieval solution, endogenous peroxidase was inactivated by incubation in 3% H2O2 for 20 min. Next, the sections were pre-incubated with 10% normal goat serum and then incubated overnight with primary antibodies panel: CD4 antibody (CST, 48274, 1:100), CD8 antibody (CST, 55336, 1:300) and CXCR6 antibody (abcam, ab273116,1:1000). Next, TMA sections were incubated with the corresponding HRP-conjugated goat anti-rabbit second antibodies (ZSBIO, CA) for 10-30 mins at room temperature. The antigenic binding sites were visualized using the OPAL dye: Opal -650 (AKOYA),Opal −520 (AKOYA), Opal- 570 (AKOYA) for each antibody, respectively. Then, TMA sections were counterstained with DAPI (Sigma) for 3-5 mins. Similar to the data analysis of immunohistochemistry, Multiplexed fluorescence staining images were analyzed and quantified by HALO software (Indica Labs, Corrales, NM, USA) as well. For the convenience of downstream comparisons, the CD4⁺CXCR6⁺, CD4⁺CXCR6^-, CD8⁺CXCR6⁺ and CD4⁺CXCR6^- were analyzed, respectively.

Li X., Gao Z., Chen J., Feng S., Luo X., Shi Y., Tang Z., Liu W., Zhang X., Huang A., Gao Q., Ke A., Zhou J., Fan J., Fu X, & Ding Z. (2023). Integrated single cell and bulk sequencing analysis identifies tumor reactive CXCR6+ CD8 T cells as a predictor of immune infiltration and immunotherapy outcomes in hepatocellular carcinoma. Frontiers in Oncology, 13, 1099385.

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Multiplex Fluorescent RNA Imaging of Fetal Gut

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Fetal gut tissue was embedded in OCT and frozen on an isopentane-dry ice slurry at −60 °C, and then cryosectioned onto SuperFrost Plus slides at a thickness of 10 μm. Before staining, tissue sections were post-fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C, then dehydrated through a series of 50%, 70%, 100% and 100% ethanol, for 5 min each. Staining with the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay (Advanced Cell Diagnostics, Bio-Techne) was automated using a Leica BOND RX, according to the manufacturers’ instructions. After manual pre-treatment, automated processing included epitope retrieval by protease digestion with Protease IV for 30 min prior to RNAscope probe hybridization and channel development with Opal 520, Opal 570, and Opal 650 dyes (Akoya Biosciences). Stained sections were imaged with a Perkin Elmer Opera Phenix High-Content Screening System, in confocal mode with 1 μm z-step size, using a 20× water-immersion objective (NA 0.16, 0.299 μm per pixel). Channels: DAPI (excitation 375 nm, emission 435–480 nm), Opal 520 (ex. 488 nm, em. 500–550 nm), Opal 570 (ex. 561 nm, em. 570–630 nm), Opal 650 (ex. 640 nm, em. 650–760 nm). The fourth channel was developed using TSA-biotin (TSA Plus Biotin Kit, Perkin Elmer) and streptavidin-conjugated Atto 425 (Sigma-Aldrich).

Elmentaite R., Kumasaka N., Roberts K., Fleming A., Dann E., King H.W., Kleshchevnikov V., Dabrowska M., Pritchard S., Bolt L., Vieira S.F., Mamanova L., Huang N., Perrone F., Goh Kai’En I., Lisgo S.N., Katan M., Leonard S., Oliver T.R., Hook C.E., Nayak K., Campos L.S., Domínguez Conde C., Stephenson E., Engelbert J., Botting R.A., Polanski K., van Dongen S., Patel M., Morgan M.D., Marioni J.C., Bayraktar O.A., Meyer K.B., He X., Barker R.A., Uhlig H.H., Mahbubani K.T., Saeb-Parsy K., Zilbauer M., Clatworthy M.R., Haniffa M., James K.R, & Teichmann S.A. (2021). Cells of the human intestinal tract mapped across space and time. Nature, 597(7875), 250-255.

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Multiplex Immunohistochemistry for Tissue Profiling

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Slides were stained in accordance with the manufactures protocol (Akoya Biosciences, Catalog #NEL821001KT). The following primary antibodies were used: CD3 (Clone Sp7, Abcam, #ab86734), CD8 (Clone C8/144B, Dako Agilent, Catalog #M710301-2), FoxP3 (Clone 236A/E7, EBioscience, Catalog #14-4777-82), CD20 (Clone L26, ThermoFisher, Catalog #14-0202-82), CD68 (Clone PG-M1, ThermoFisher, Catalog #MA5-12407), CD163 (Clone 10D6, ThermoFisher, Catalog # MA5-11458), Ki-67 (Clone MIB-1, Dako, Catalog #M724029-2), CD14 (Clone EPR3653, Abcam, Catalog #ab133335), PanCK (Clone AE1/AE3 + 5D3, Abcam, Catalog #ab86734), in combination with Opal Polaris 480 (Akoya Biosciences, Catalog #FP1500001KT), Opal Polaris 780 (Akoya Biosciences, Catalog #FP1501001KT) and Opal 520, Opal 540, Opal 570, Opal 620, Opal 650 and Opal 690 (Akoya Biosciences, Catalog #NEL821001KT). Slides were imaged using the Vectra Polaris.

Zwart E.S., van Ee T., Affandi A.J., Boyd L.N., Rodriguez E., den Haan J.M., Farina A., van Grieken N.C., Meijer L.L., van Kooyk Y., Mebius R.E, & Kazemier G. (2023). Spatial immune composition of tumor microenvironment in patients with pancreatic cancer. Cancer Immunology, Immunotherapy : CII, 72(12), 4385-4397.

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Multiparametric analysis of skin biomarkers

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FFPE skin blocks were cut into 6 μm sections and placed on slides. Sections were first deparaffinized and rehydrated, then Heat-Induced Epitope Retrieval (HIER) was performed and sections were permeabilized with PBS 0.01% Triton. Samples were stained for 2 h at room temperature with the following primary antibodies: goat anti-human IL-26/AK155 (R&D systems, #AF1375, 1/100), mouse anti-human TGF beta-1 (Thermo Fisher Scientific, # MA5-16949, 1/500), rabbit anti-human CD3 (Ventana, # 790-4341, ready to use). Sections were then stained for 30 min at room temperature with the following fluorescently-labeled secondary antibodies: donkey anti-rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen, #A-32790, 1/500), chicken anti-mouse IgG (H + L) Alexa Fluor 647 (Invitrogen, #A-21463, 1/500), donkey anti-goat IgG (H + L) Alexa Fluor 546 (Invitrogen, #A-32790, 1/500). For RNA ISH, TGFB1 mRNA (#400881) was detected in skin using RNAScope® Multiplex Fluorescent V2 Assay following the manufacturer’s instruction (Advanced Cell Diagnostics, Inc). Sections were then labeled with OPAL 650 (Akoya Biosciences, #OP-001005, 1/1500). All slides were mounted with ProLong Gold antifade mounting with DAPI (Thermo Fisher Scientific). Images were acquired with a Zeiss LSM 700 confocal microscope and analyzed with Zen 2010 software.

Fries A., Saidoune F., Kuonen F., Dupanloup I., Fournier N., Guerra de Souza A.C., Haniffa M., Ma F., Gudjonsson J.E., Roesner L., Li Y., Werfel T., Conrad C., Gottardo R., Modlin R.L., Di Domizio J, & Gilliet M. (2023). Differentiation of IL-26+ TH17 intermediates into IL-17A producers via epithelial crosstalk in psoriasis. Nature Communications, 14, 3878.

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Multiplex Immunofluorescence Tissue Imaging

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Multiplex immunofluorescence (IF) staining, validation and analysis are detailed in Supplementary Methods and Supplementary Figs. S2 and S3. Briefly, 4-μm sections of formalin-fixed paraffin-embedded (FFPE) tissue samples were deparaffinised and antigen retrieval was performed using DAKO PT-Link heat-induced antigen retrieval with a low- (pH 6) or high-pH (pH 9) solution (DAKO). Samples were stained with mAbs targeting cytokeratin (CK), CD4, CD8, FOXP3, PD-1 and CD137 followed by TSA visualisation with fluorophores Opal 520, Opal 540, Opal 570, Opal 620, Opal 650 and Opal 690 (Akoya Biosciences), as described earlier.²⁰ Each tissue section was put through several sequential rounds of antibody staining. In the seventh round, nuclei were counterstained with spectral DAPI (Akoya Biosciences) and sections mounted with Faramount Aqueous Mounting Medium (Dako). Multiplexed immunofluorescence slides were scanned on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Tissue imaging, spectral unmixing and phenotyping were performed using inForm software (version 2.4.8, Akoya Biosciences), as described previously.²⁰

Salas-Benito D., Conde E., Tamayo-Uria I., Mancheño U., Elizalde E., Garcia-Ros D., Aramendia J.M., Muruzabal J.C., Alcaide J., Guillen-Grima F., Minguez J.A., Amores-Tirado J., Gonzalez-Martin A., Sarobe P., Lasarte J.J., Ponz-Sarvise M., De Andrea C.E, & Hervas-Stubbs S. (2021). The mutational load and a T-cell inflamed tumour phenotype identify ovarian cancer patients rendering tumour-reactive T cells from PD-1+ tumour-infiltrating lymphocytes. British Journal of Cancer, 124(6), 1138-1149.

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Multiplex Immunofluorescence Staining and Analysis

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Fluorescence IHC staining, imaging, and analysis were performed as described previously (57 (link)), where rabbit anti-IDO1 (SP260, 1:500 dilution, Abcam) was detected with Opal 620 (1:200 dilution, Akoya Biosciences) followed by chemical quenching of residual HRP using 100 mM benzhydrazide with 50 mM hydrogen peroxide. Mouse anti–HLA-DR (TAL.1B5, 1:500 dilution, Agilent) was detected with Opal 650 (1:200 dilution, Akoya Biosciences), and then all primary and secondary antibodies were removed via microwave to allow staining with rabbit anti-CD11b (EY1345Y, 1:500 dilution, Abcam) detected with Opal 520 (1:200 dilution, Akoya Biosciences) followed by chemical quenching of residual HRP using 100 mM benzhydrazide with 50 mM hydrogen peroxide. Finally, mouse anti–pan-cytokeratin (AE1/AE3, 1:100, Agilent) was detected with Opal 570 (1:200 dilution, Akoya Biosciences), and DAPI was used to identify cell nuclei. Image analysis was performed using AQUA (Navigate BioPharma Services, Inc.) to determine the percentage of all DAPI⁺ cells that are CD11b⁺, HLA-DR⁺, IDO1⁺, CD11b⁺HLA-DR⁺, or CD11b⁺HLA-DR^– cells. The IDO1 channel was not used for analysis in the present study.

Franklin D.A., Sharick J.T., Ericsson-Gonzalez P.I., Sanchez V., Dean P.T., Opalenik S.R., Cairo S., Judde J.G., Lewis M.T., Chang J.C., Sanders M.E., Cook R.S., Skala M.C., Bordeaux J., Orozco Bender J., Vaupel C., Geiss G., Hinerfeld D, & Balko J.M. (2020). MEK activation modulates glycolysis and supports suppressive myeloid cells in TNBC. JCI Insight, 5(15), e134290.

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