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Opal 520

Manufactured by Akoya Biosciences
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The Opal 520 is a fluorescent reagent for use in multiplex immunohistochemistry (IHC) and immunofluorescence (IF) applications. It is designed to provide a bright, stable fluorescent signal for the detection of protein targets in tissue samples.

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Lab products found in correlation

Opal 570, by Akoya Biosciences (27 mentions) Opal 690, by Akoya Biosciences (16 mentions) Opal 620, by Akoya Biosciences (11 mentions) Opal 650, by Akoya Biosciences (9 mentions) Rnascope multiplex fluorescent reagent kit v2 assay, by Advanced Cell Diagnostics (5 mentions) Bond rx, by Leica (4 mentions) Lsm800 confocal microscope, by Zeiss (4 mentions) Lsm 710, by Zeiss (4 mentions) Vectra polaris, by Akoya Biosciences (3 mentions) Opal polymer horseradish peroxidase, by Akoya Biosciences (3 mentions) Rnascope multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (3 mentions) Atto 425, by Merck Group (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Hg 4000 012, by Epredia (2 mentions) Axio zoom v16 microscope, by Zeiss (2 mentions) Plan apochromat 63x 1.4 na oil objective, by Zeiss (2 mentions) Mm per2 c1 and mm arntl c2 rna probes, by Akoya Biosciences (2 mentions) Opal 570 dye, by Akoya Biosciences (2 mentions) Rnascope multiplex fluorescent assay, by Advanced Cell Diagnostics (2 mentions) Vt1200, by Leica (2 mentions)

47 protocols using opal 520

1

Multicolor RNA Expression Profiling

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RNA-scope experiment was performed following the manual of RNAscope multiplex fluorescent reagent kit V2 assay (Advanced Cell Diagnostics). Briefly, embryo sections were post-fixed with 4% PFA for 15 min, followed by sequential dehydration in 50%, 70%, and 100% ethanol. Sections were then treated with hydrogen peroxide for 10 min at room temperature. Antigen retrieval was performed at 99 °C for 5 min in 1× Target Retrieval Reagent. Sections were then digested with Protease III for 30 min at 40 °C. For probe hybridization, probes (EYFP-C1; Cat# 312131, Mm-Thy1-C2; Cat# 430661-C2 and Mm-Pdgfra-C3; 480661-C3) were diluted according to manufacturer’s instructions and incubated with sections for 120 min at 40 °C. After washing, signal amplification was conducted by sequential hybridization with AMP1 (30 min), AMP2 (30 min), and AMP3 (15 min). For signal development, Opal 520, Opal 620, and Opal 690 (Akoya Biosciences) were diluted at 1:1500 and assigned to C1, C2, and C3, respectively.
Fung C.W., Zhou S., Zhu H., Wei X., Wu Z, & Wu A.R. (2022). Cell fate determining molecular switches and signaling pathways in Pax7-expressing somitic mesoderm. Cell Discovery, 8, 61.
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2

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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3

Olfactory Brain Structure Analysis via RNAscope

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We dissected the brain of P14–21 C57BL/6NCrl mice at ZT3.5 or ZT15.5, removed the olfactory bulbs, cerebellum and temporal lobes and fixed it with 4% PFA/PBS overnight at 4°C. The brain was then cryo-protected in 30% sucrose PBS at 4°C for 48 hour and stored in PBS for no more than a week. To prepare slices for RNAscope, we separated the two hemispheres, embedded them in agar and prepared 40 μm thick slices using a vibrating blade microtome (VT1200S, Leica Microsystems, Buffalo Grove, IL). The slices were post-fixed in 4% PFA/PBS for 30 min at room temperature (RT) and mounted onto Superfrost plus microscope slides. Mounted slices were used for fluorescence in situ hybridization (FISH) using an RNAscope multiplex fluorescent assay (Advanced Cell Diagnostics, Newark, CA) according to manufacturer instructions, using Mm-Per2-C1 and Mm-Arntl-C2 RNA probes and Opal 520 and Opal 570 dyes (Akoya Biosciences, Menlo Park, CA). DAPI Fluoromount G was used as the mounting medium (Cat# 0100–02; SouthernBiotech, Birmingham, AL). The presence of Per2 and Arntl transcripts was assessed using a confocal microscope (Zeiss LSM710) equipped with a Plan-Apochromat 63X/1.4NA oil objective. Image size was set to 1024×1024 pixels and represented the average of 8 consecutive frames.
McCauley J.P., Petroccione M.A., D’Brant L.Y., Todd G.C., Affinnih N., Wisnoski J.J., Zahid S., Shree S., Sousa A.A., De Guzman R.M., Migliore R., Brazhe A., Leapman R.D., Khmaladze A., Semyanov A., Zuloaga D.G., Migliore M, & Scimemi A. (2020). Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1. Cell reports, 33(2), 108255.
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4

Multiplex FISH Analysis of Neuronal Markers

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Fluorescent in situ hybridization reactions were performed using an RNAscope Multiplex Fluorescent Reagent Kit v2 (ACDBio, #323100) according to the manufacturer’s instructions. Brain sections mounted onto SuperFrost Plus glass slides (VWR, #48311–703) were labeled using a combination of target probes for Crh (Probe-Mm-Crh, #316091), Tac1 (Probe-Mm-Tac1, #410351; or Probe-Mm-Tac1-C3, #410351-C3), Slc17a6 (Probe-Mm-Slc17a6-C2, #319171-C2), Calb1 (Probe-Mm-Calb1-C2, #428431-C2), Calb2 (Probe-Mm-Calb2-C2, #313641-C2), Pvalb (Probe-Mm-Pvalb-C2, #421931-C2), and Fos (Probe-Mm-Fos-C2; #316921-C2). The Fos probe was diluted 1:10 to reduce background hybridization. Fluorophore reagent packs (Akoya Biosciences) including Opal 520 (FP1487001KT), Opal 570 (FP1488001KT), and Opal 690 (FP1497001KT) were diluted to a final concentration of 1:1,500 with TSA buffer. Following staining procedures, slides were coverslipped with Fluoromount-G (Southern Biotech, #0100–01) and stored in the dark at 4 °C until microscopy and imaging.
Kim J.H., Kromm G.H., Barnhill O.K., Sperber J., Heuer L.B., Loomis S., Newman M.C., Han K., Gulamali F.F., Legan T.B., Jensen K.E., Funderburk S.C., Krashes M.J, & Carter M.E. (2022). A discrete parasubthalamic nucleus subpopulation plays a critical role in appetite suppression. eLife, 11, e75470.
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5

Olfactory Brain Structure Analysis via RNAscope

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We dissected the brain of P14–21 C57BL/6NCrl mice at ZT3.5 or ZT15.5, removed the olfactory bulbs, cerebellum and temporal lobes and fixed it with 4% PFA/PBS overnight at 4°C. The brain was then cryo-protected in 30% sucrose PBS at 4°C for 48 hour and stored in PBS for no more than a week. To prepare slices for RNAscope, we separated the two hemispheres, embedded them in agar and prepared 40 μm thick slices using a vibrating blade microtome (VT1200S, Leica Microsystems, Buffalo Grove, IL). The slices were post-fixed in 4% PFA/PBS for 30 min at room temperature (RT) and mounted onto Superfrost plus microscope slides. Mounted slices were used for fluorescence in situ hybridization (FISH) using an RNAscope multiplex fluorescent assay (Advanced Cell Diagnostics, Newark, CA) according to manufacturer instructions, using Mm-Per2-C1 and Mm-Arntl-C2 RNA probes and Opal 520 and Opal 570 dyes (Akoya Biosciences, Menlo Park, CA). DAPI Fluoromount G was used as the mounting medium (Cat# 0100–02; SouthernBiotech, Birmingham, AL). The presence of Per2 and Arntl transcripts was assessed using a confocal microscope (Zeiss LSM710) equipped with a Plan-Apochromat 63X/1.4NA oil objective. Image size was set to 1024×1024 pixels and represented the average of 8 consecutive frames.
McCauley J.P., Petroccione M.A., D’Brant L.Y., Todd G.C., Affinnih N., Wisnoski J.J., Zahid S., Shree S., Sousa A.A., De Guzman R.M., Migliore R., Brazhe A., Leapman R.D., Khmaladze A., Semyanov A., Zuloaga D.G., Migliore M, & Scimemi A. (2020). Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1. Cell reports, 33(2), 108255.
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6

Fluorescent In Situ Hybridization of Tissue

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In situ hybridizations were performed on freshly sectioned cryo-embedded tissue samples. Tissue sections (14 μm) were prepared as described above. RNA in situ hybridization was performed using the RNA scope system from Advanced Cell Diagnostics. Probes for mouse Gli1 and Vegf-A were also purchased from ACD, as were washing buffer and retrieval reagents. For fluorescence detection, the following Opal dyes were purchased by Akoya Biosciences: Opal 520 (Cat # FP1487001KT), Opal 570 (Cat # FP1488001KT), Opal 620 (Cat # FP1497001KT), Opal 690 (Cat # FP1495001KT)
Faniku C., Kong W., He L., Zhang M., Lilly G, & Pepper J. (2020). Hedgehog signaling promotes endoneurial fibroblast migration and Vegf-A expression following facial nerve injury. Brain research, 1751, 147204.
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7

Single-Molecule RNA FISH in Mouse Embryos

Cited 1 time
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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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8

Multiplexed IHC Analysis of COVID-19 Lung Tissue

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Multiplexed IHC was performed with a Leica Bond Rx on 5 μm-thick formalin-fixed paraffin-embedded lung tissue sections from individuals with and without COVID-19. Consecutive staining was performed by heat-induced antigen retrieval followed by incubation with primary antibody (anti-C5aR1 clone S5/1 at 1 μg/mL). The signal was amplified and detected with Opal™ polymer horseradish peroxidase and Opal 520 (Akoya Biosciences). The sections were then subjected to heat-induced antibody stripping and incubated with the next antibody (anti-CD163 clone EDHu-1 at 1 μg/mL, detected with Opal 620, and, finally, anti-CD68 clone KP1 at 0.1 μg/mL, detected with Opal 690) and spectral DAPI. All Opal reagents were used at a dilution of 1/150. Slides were finally mounted in ProLong Diamond antifade mounting medium (Thermo Fisher) and scanned with a Vectra Polaris (Akoya Biosciences). Hematoxylin and eosin-stained slides were scanned with a Nanozoomer (Hamamatsu). After spectral deconvolution and whole-slide reconstruction of the multiplexed IHC stained sections, digital pathology methods were used to determine the density of positive cells. All analyses were performed with Halo (Indica Labs) and R.
Carvelli J., Demaria O., Vély F., Batista L., Benmansour N.C., Fares J., Carpentier S., Thibult M.L., Morel A., Remark R., André P., Represa A., Piperoglou C., Cordier P.Y., Le Dault E., Guervilly C., Simeone P., Gainnier M., Morel Y., Ebbo M., Schleinitz N, & Vivier E. (2020). Association of COVID-19 inflammation with activation of the C5a-C5aR1 axis. Nature, 588(7836), 146-150.
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9

Multiplex Fluorescent RNA-ISH with RNAscope

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For RNA-ISH, the RNAscope® system (Advanced Cell Technologies, Worcester, MA, USA) was applied according to the manufacturer’s protocol. In brief, tissue sections were prepared as described below (Immunofluorescence staining). After dehydration, HRP was quenched with Bloxall (Sp-6000, Vector Technologies, Stuttgart, Germany) blocking solution for 10 min at RT followed by Pretreat III solution for 30 min at RT. Then, RNAscope®probes (Supplementary Table S3) were hybridized on the sections for 2 h at 40 °C, and then underwent an RNAscope® multiplex fluorescent Reagent kit v2 assay according to the manufacturer’s instructions. Opal 520, Opal 570, and Opal 690 (Akoya Biosciences, Marlborough, MA, USA) were diluted 1:1000. Sections were mounted with ProLong®Gold mounting medium, and images were obtained using a confocal microscope (Leica Microsystems Sp8, Wetzlar, Germany) at 400×.
Zhou A.X., Jeansson M., He L., Wigge L., Tonelius P., Tati R., Cederblad L., Muhl L., Uhrbom M., Liu J., Björnson Granqvist A., Lerman L.O., Betsholtz C, & Hansen P.B. (2024). Renal Endothelial Single-Cell Transcriptomics Reveals Spatiotemporal Regulation and Divergent Roles of Differential Gene Transcription and Alternative Splicing in Murine Diabetic Nephropathy. International Journal of Molecular Sciences, 25(8), 4320.
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10

Multiplexed RNA Visualization in Mouse Brain

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Mouse Myrf mRNA probe (ACDbio, Cat# 524061), mouse Mbp mRNA probe (ACDbio, Cat# 451491) and RNAscope Multiplex Fluorescent Reagent Kit V2 assay (ACDbio, Cat# 323110) were purchased from ACDbio company. Fluorophores were purchased from Akoya biosciences (Opal 520: Cat# FP1487001KT, Opal 620: Cat# FP1495001KT). Mice were processed for RNAscope as follows. Deeply anesthetized animals were transcardially perfused with 0.9% saline followed by ice-cold 4% PFA as described above. Brains were immediately dissected out and transferred to 10% NB formalin solution (Sigma, Cat# HT5011) at RT for exactly 24 hours. Brains were then transferred to freshly made 70% ethanol for 24 hours at RT and processed for paraffin embedding. Sections were cut at 5 mm. RNAscope assay was performed as per manufacturer’s specifications.
Xu H., Dzhashiashvili Y., Shah A., Kunjamma R.B., Weng Y.L., Elbaz B., Fei Q., Jones J.S., Li Y.I., Zhuang X., Ming G.L., He C, & Popko B. (2019). m6A mRNA methylation is essential for oligodendrocyte maturation and CNS myelination. Neuron, 105(2), 293-309.e5.
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