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Opal 570

Manufactured by Akoya Biosciences
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The Opal 570 is a multiplex immunofluorescence imaging system developed by Akoya Biosciences. The core function of the Opal 570 is to enable the simultaneous detection and visualization of multiple protein targets within a single tissue sample.

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Lab products found in correlation

Opal 520, by Akoya Biosciences (27 mentions) Opal 690, by Akoya Biosciences (19 mentions) Opal 620, by Akoya Biosciences (11 mentions) Opal 650, by Akoya Biosciences (10 mentions) Lsm 880 confocal microscope, by Zeiss (6 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (4 mentions) Lsm800 confocal microscope, by Zeiss (4 mentions) Multiplex fluorescent v2 kit, by Advanced Cell Diagnostics (3 mentions) Opal dyes, by Akoya Biosciences (3 mentions) Atto 425, by Merck Group (3 mentions) Rnascope multiplex fluorescent reagent kit v2 assay, by Advanced Cell Diagnostics (3 mentions) Hg 4000 012, by Epredia (2 mentions) Axio zoom v16 microscope, by Zeiss (2 mentions) Ab64269, by Abcam (2 mentions) E0432, by Abcam (2 mentions) Fluoromount g, by Southern Biotech (2 mentions) Mounting media, by Vector Laboratories (2 mentions) Vectra polaris automated quantitative pathology imaging system, by Akoya Biosciences (2 mentions) Rnascope multiplex fluorescent reagent kit v2, by Advanced Cell Diagnostics (2 mentions) Tsa plus biotin kit, by PerkinElmer (2 mentions)

45 protocols using opal 570

1

Multiplex RNA Profiling of FFPE Tissues

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Sections were cut from FFPE blocks at a thickness of 5 μm using a Leica microtome, placed onto SuperFrost Plus slides (VWR), and baked overnight at 55°C to dry and ensure adhesion. Tissue sections were then processed using a Leica BOND RX to automate staining with the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay (Advanced Cell Diagnostics, Bio-Techne), according to the manufacturers' instructions. Automated processing included baking at 60°C for 30 minutes and dewaxing, as well as heat-induced epitope retrieval at 95°C for 15 minutes in buffer ER2 and digestion with Protease III for 15 minutes.
For visualising markers prior to NanoString GeoMX profiling, 3-plex RNAscope was developed using tyramide signal amplification with Opal 570, Opal 620, and Opal 690 dyes (Akoya Biosciences). No nuclear stain was applied at this stage.
For validation staining, 3-plex or 4-plex RNAscope stains were developed using Opal 520, Opal 570, and Opal 650 dyes (Akoya Biosciences), as well as TSA-biotin and streptavidinconjugated Atto 425 (Sigma). Nuclei were counterstained with DAPI at 167 ng/ml.
Roberts K., Aivazidis A., Kleshchevnikov V., Li T., Fropf R., Rhodes M., Beechem J., Hemberg M., & Bayraktar O.A. (2021). Transcriptome-wide spatial RNA profiling maps the cellular architecture of the developing human neocortex.
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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

Quantitative Analysis of PTPN23 and EGFR Expression

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Expression of PTPN23 was analyzed by RNAscope multiplex fluorescence assay (Advance cell diagnostics, ACD, a BioTechne brand) following the manufacturer’s instructions. RNAscope was used for PTPN23 as it provides a very accurate and quantitative assessment of gene expression levels. PTPN23 targets the availability of EGFR protein. Therefore, EGFR protein expression was analyzed using immunofluorescence to focus on the spatial distribution of this receptor. Sections were dewaxed 3 × 10 min in xylene and rehydrated in ethanol series (100%, 90%, 70%, 50%, and 30%) for a period of 2 min for each step. Antigen retrieval was performed in citric acid 0.01 M at 90 °C for 30 min or Tris EDTA at 90 °C for 30 min. Blocking was achieved in PBS with 0.025% Tween20, 1% BSA, and 10% serum. Primary antibody (anti-pEGFR) was applied with an overnight incubation at 4 °C. Secondary Goat anti-Rabbit biotin was used 1/800 (Dako, E0432) followed by Streptavidin–HRP (Abcam, ab64269). The color reaction was performed in TSA buffer (100 mM Borate buffer with 0.0003% hydrogen peroxidase) with Opal-570 1/300 (Akoya Bioscience, OP001003) for 10 min. Slides were mounted with fluoroshield with DAPI as a nuclei stain.
Adisornkanj P., Chanprasit R., Eliason S., Fons J.M., Intachai W., Tongsima S., Olsen B., Arold S.T., Ngamphiw C., Amendt B.A., Tucker A.S, & Kantaputra P. (2023). Genetic Variants in Protein Tyrosine Phosphatase Non-Receptor Type 23 Are Responsible for Mesiodens Formation. Biology, 12(3), 393.
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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

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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6

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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7

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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8

Multicolor Immunofluorescence Staining Protocol

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Sections were dewaxed 3 × 10 min in xylene and rehydrated in EtOH series (100%, 90%, 70%, 50%, and 30%) for a period of 2 min for each step. Antigen retrieval was performed in citric acid 0.01M at 90°C for 30 min. Blocking was achieved in PBS with 0.025% Tween20, 1% BSA, and 10% serum. After ON incubation at 4°C, the primary antibody was washed in PBT 0.025%, and a secondary antibody was incubated for 1 h at RT. After washing, the slides were mounted with fluoroshield. Primary antibodies were anti-acetylated tubulin 1/300 (Sigma-Aldrich, T7451), anti-Plunc1 1/200 (R&D systems, AF4274), and anti-K5 1/300 (Biolegend, PRB-160P), and secondaries were Goat anti-Mouse-568 1/500 (Invitrogen, A11004), Donkey anti-Sheep-488 1/500 (Invitrogen, A11015), and Donkey anti-Rabbit 647 1/500 (Invitrogen, A31573).
For the detection of PCNA 1/300 (Abcam, ab19166) and P63 1/400 (Abcam, ab124762), a secondary Goat anti-Rabbit biotin was used 1/800 (Dako, E0432) followed by Streptavidin-HRP (Abcam, ab64269). The color reaction was performed in TSA buffer (100 mM Borate buffer with 0.0003% hydrogen peroxidase) with Opal-570 1/300 (Akoya Bioscience, OP-001003) for 10min. DAPI was used to stain nuclei. For each genotype, N = 3.
Fons J.M., Milmoe N.J., Dack M.R., Joshi L., Thompson H, & Tucker A.S. (2022). The interconnected relationships between middle ear bulla size, cavitation defects, and chronic otitis media revealed in a syndromic mouse model. Frontiers in Genetics, 13, 933416.
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9

Multiplex In-Situ Hybridization for FGF21 and tdTomato

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Mice were euthanized and brain tissue was collected, frozen in 2-methylbutane, embedded into medium (Tissue-Tek O.C.T. Compound) and stored at −80°C. 10-μm coronal sections were cut using a cryostat and stored at −80°C until use. Tissue sections underwent treatment and hybridization according to manufacturer (Advanced Cell Diagnostics) instructions for fresh frozen tissue (Multiplex Fluorescent Reagent Kit v2 User Manual). Briefly, slides were transferred from −80°C directly into 4% paraformaldehyde at 4°C. Slides were rinsed and dehydrated in increasing ethanol concentrations before treatment with hydrogen peroxide and pro-tease IV at room temperature. Slides were subsequently moved to 40°C (HybEZ oven; ACD) for hybridization with probes targeting FGF21 (460931) and tdTomato (317041-C2), as well as positive (Mm-Ppib; 313911) and negative (DapB; 310043) control probes. The hybridization signal was amplified, developed, and subsequently incubated with fluorescent probes Opal 520 and Opal 570 (Akoya Biosciences). After every incubation step, slides were washed in 1X Wash Buffer three times (2 min each). Slides were coverslipped with mounting media (VECTASHIELD) and imaged with a Leica STED.
Zhou B., Claflin K.E., Flippo K.H., Sullivan A.I., Asghari A., Tadinada S.M., Jensen-Cody S.O., Abel T, & Potthoff M.J. (2022). Central FGF21 production regulates memory but not peripheral metabolism. Cell reports, 40(8), 111239.
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10

In situ Hybridization of MAF Transcription Factors

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In situ hybridization was performed by RNAscope technology using the Fluorescent Multiplex Assay kit v2 (Advanced Cell Diagnostics (ACD)). OB and cerebral cortex were sectioned using a cryostat into 14 μm slices and stored at −80 °C until use. Sections were pretreated according to the manufacturer’s protocol and hybridized with probes specific for mafa, mafb, c-maf, vglut1, and gad65 at 40 °C for 2 hours. After hybridization, the signals were amplified and fluorescently developed with Opal 520 and Opal 570 (1:1500; Akoya Biosciences; Marlborough, MA). The sections were counterstained with DAPI for 30 sec and mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific). All the probes were designed by ACD and purchased from them (mafa: Cat# 556931; mafb: Cat# 438531-C2, c-maf: Cat# 412951; vglut1: Cat# 416631-C3; and gad65: Cat# 439371-C3).
Ito A, & Imamura F. (2021). Expression of Maf family proteins in glutamatergic neurons of the mouse olfactory bulb. Developmental neurobiology, 82(1), 77-87.
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