Spectral dapi

Manufactured by Akoya Biosciences

Sourced in United States

Spectral DAPI is a fluorescent DNA-binding dye used for labeling and visualizing nucleic acids in biological samples. It is a core product offered by Akoya Biosciences for research applications.

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Spectral DAPI (4′,6-diamidino-2-phenylindole, (3 citations) Spectral DAPI solution (2 citations)

26 protocols using spectral dapi

Multiplex Immunofluorescence Analysis of NK Cell Markers

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Formalin-fixed, paraffin-embedded slides were stained for NKp46 (rabbit anti-human NKp46, Abcam, clone EPR22403-57), CD57 (mouse anti-human CD57, Thermo Scientific, clone NK1), and CD94 (rabbit anti-human CD94, Abcam, clone EPR21003) expression. Immunofluorescence (IF) was evaluated using NKp46 and 4′,6-diamidino-2-phenylindole (DAPI). Immunohistochemical expression was scored by a blinded pathologist (MD) as described previously.25 (link) IF staining was performed using Opal fluorophores and counterstained with Spectral DAPI (Akoya Biosciences). The Ventana Discovery Ultra system was used for image acquisition, and data analysis was performed as described previously.33 (link)

Perry L.M., Cruz S.M., Kleber K.T., Judge S.J., Darrow M.A., Jones L.B., Basmaci U.N., Joshi N., Settles M.L., Durbin-Johnson B.P., Gingrich A.A., Monjazeb A.M., Carr-Ascher J., Thorpe S.W., Murphy W.J., Eisen J.A, & Canter R.J. (2023). Human soft tissue sarcomas harbor an intratumoral viral microbiome which is linked with natural killer cell infiltrate and prognosis. Journal for Immunotherapy of Cancer, 11(1), e004285.

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Automated Multiplex Immunofluorescence Staining

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The validation pipeline and details of the development of the multiplex immunolabeling protocol have been described previously by our group and are shown in supplementary material, Figure S1 and Supplementary materials and methods [15 (link), 16 (link)]. Multiplex immunofluorescence staining was performed using the LabSat® Research platform (Lunaphore Technologies, Tolochenaz, Switzerland), a fully automated tissue‐staining instrument for rapid immunostaining which utilizes a microfluidic technology for the rapid and uniform delivery of reagents to tissue samples [17 (link)]. In brief, each TMA section was subjected to six successive, automated rounds of antibody staining, including pan‐cytokeratin, CD8, CD68, FOXP3, PD‐1, and PD‐L1. Nuclei were counterstained with spectral DAPI (Akoya Biosciences, Marlborough, MA, USA). A full protocol is provided in Supplementary materials and methods. TMAs were scanned using a PhenoImager HT Automated Quantitative Pathology Imaging System (Akoya Biosciences). Image analysis was performed using the inForm software framework (version 2.4.8, Akoya Biosciences), shown in Supplementary materials and methods.

López‐Janeiro Á., Villalba‐Esparza M., Brizzi M.E., Jiménez‐Sánchez D., Ruz‐Caracuel I., Kadioglu E., Masetto I., Goubert V., Garcia‐Ros D., Melero I., Peláez‐García A., Hardisson D, & de Andrea C.E. (2022). The association between the tumor immune microenvironments and clinical outcome in low‐grade, early‐stage endometrial cancer patients. The Journal of Pathology, 258(4), 426-436.

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Automated Tissue Immunofluorescence Imaging

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Immunofluorescence (IF) was performed at HistoWiz Inc. following a standard operating procedure under GLP regulations and a fully automated workflow. Mouse tissue was sectioned (4 μm) transversely to the orientation of muscle fibers and mounted on positively charged slides. DAPI counterstaining to visualize nuclei was performed on the Bond Rx Auto Stainer (Leica Biosystems, Wetzlar, Germany) using Spectral Dapi (Akoya FP1490, Akoya Biosciences, Marlborough, MA, USA). Following staining, slides were cover slipped with Prolong Diamond Antifade Mounting Medium (Thermo P36961) to prevent bleaching and signal loss. Finally, slides were imaged with a DAPI filter and a Cy5-MSI filter aligned to capture A647. Gain and exposure were kept consistent across all slides with DAPI at 0.72 ms and 6.2 ms for Cy5-MSI.

Maji D., Miguela V., Cameron A.D., Campbell D.A., Sasset L., Yao X., Thompson A.T., Sussman C., Yang D., Miller R., Drozdz M.M, & Liberatore R.A. (2024). Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies. Pharmaceutics, 16(4), 547.

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Tyramide Signal Amplification for Multiplex Immunofluorescence

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The Tyramide Signal Amplification (TSA)-based Opal method (Akoya Biosciences) was used for mIF staining on the Leica BOND RX automated immunostainer (Leica Microsystems). Prior to staining, all 4 µm-thick FFPE tissue sections were deparaffinised by baking overnight at 56 °C, soaking in BOND Dewax Solution at 72 °C, and then rehydrating in ethanol. Heat-induced epitope retrieval (HIER) pretreatments were applied using BOND Epitope Retrieval (ER) Solutions citrate-based pH 6.0 ER1 or EDTA-based pH 9.0 ER2 (both Leica Biosystems). Tissue sections were blocked with Normal Goat Serum (Vector Laboratories) for 10 min before applying each primary antibody. A fluorescent singleplex was carried out for melanoma cells biomarker to determine the optimal staining conditions. The rabbit anti-mouse Melan-a (Abcam, clone EPR20380) primary antibody was subsequently added on the slides. The HRP-conjugated secondary antibodies goat anti-rabbit (Vector Laboratories) were incubated as appropriated for 10 min. The TSA-conjugated fluorophore was then added for 10 min. Slides were rinsed with washing buffer after each step. Finally, the spectral DAPI (Akoya Biosciences) was used as nuclear counterstain, and slides were mounted in ProLong Diamond Anti-fade Mountant (Life Technologies).

Muccioli S., Brillo V., Varanita T., Rossin F., Zaltron E., Velle A., Alessio G., Angi B., Severin F., Tosi A., D’Eletto M., Occhigrossi L., Falasca L., Checchetto V., Ciaccio R., Fascì A., Chieregato L., Rebelo A.P., Giacomello M., Rosato A., Szabò I., Romualdi C., Piacentini M, & Leanza L. (2023). Transglutaminase Type 2-MITF axis regulates phenotype switching in skin cutaneous melanoma. Cell Death & Disease, 14(10), 704.

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Multicolor IHC Analysis of FFPE Ileum

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FFPE blocks of ileum from controls and Crohn’s disease patients were cut into 4-μm sections and mounted on Superfrost Plus microscope slides [Thermo Fisher Scientific] at the Clinical Pathology unit, Sahlgrenska University Hospital. Slides were deparaffinized with xylene and rehydrated with successive baths of ethanol and water. Antigens were unmasked using a 2100 Antigen Retriever [Aptum Biologics]. Slides were then sequentially stained with antibodies against TREM-1, CD163, CD11c and Pan-CK [Supplementary Table 1] using an Opal 7-Color Manual IHC Kit [Akoya Biosciences] according to the manufacturer’s recommendations. Subsequently, cell nuclei were stained with spectral DAPI [Akoya Biosciences] and slides were mounted with ProLong Glass Antifade Mountant [Thermo Fisher Scientific]. Stained sections were scanned with a Metasystem automated slide scanner [MetaSystems, Germany] equipped with SpectraSplit filter system for extended multicolour imaging [Kromnigon] and a Carl Zeiss AxioImager.Z2 microscope [Carl Zeiss]. Image analysis was performed with Zeiss ZEN 3.1 software [Carl Zeiss].

Caër C., Gorreja F., Forsskåhl S.K., Brynjolfsson S.F., Szeponik L., Magnusson M.K., Börjesson L.G., Block M., Bexe-Lindskog E, & Wick M.J. (2021). TREM-1+ Macrophages Define a Pathogenic Cell Subset in the Intestine of Crohn’s Disease Patients. Journal of Crohn's & Colitis, 15(8), 1346-1361.

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Multiplex Immunohistochemistry of HNSCC Tumors

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Ten FFPE tissue of HNSCC tumors were sectioned to 4 µm thick for the subsequent multiplex immunohistochemistry via the OPAL Polaris system (Akoya Biosciences). After deparaffinization and hydration, the FFPE slides were manually stained with the CD8 (clone C8/144B, CST, #70306S), PD-1 (clone D7D5W, CST, #84,651T), anti-Ki67 (clone SP6, Abcam, #ab16667), CDK4 (clone D9G3E, CST, #12790), CD24 (10600-1-AP, Proteintech, #10600-1-AP), Anti-EPCAM (clone EPR20532-225, Abcam, #ab223582), and Anti-HLA-DR (clone EPR3692, Abcam, #ab92511) antibodies. The sections were counterstained with spectral DAPI (Akoya Biosciences). The stained slides were imaged and scanned using the Vectra Polaris multispectral imaging system.

Cheng D., Qiu K., Rao Y., Mao M., Li L., Wang Y., Song Y., Chen J., Yi X., Shao X., Huang S.H., Zhang Y., Chen X., Wu S., Yu S., Liu J., Wang H., Peng X., Li D., Yang L., Chen L., Ying Z., Zheng Y., Zheng M., Ying B., Zeng X., Zhang W., Xu W., Liu G., Chen F., Yu H., Zhao Y, & Ren J. (2023). Proliferative exhausted CD8+ T cells exacerbate long-lasting anti-tumor effects in human papillomavirus-positive head and neck squamous cell carcinoma. eLife, 12, e82705.

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Multiplex Immunofluorescence for HNSCC

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Twenty-three formalin-fixed paraffin-embedded human HNSCC samples were stained using the Opal 7-Color Automation IHC Kit-50 Slide according to manufacturer’s instructions (Akoya Biosciences) using the following antibodies and antigen retrieval processes: with antibodies against PD-L1 (clone E1L3N, Cell Signaling Technologies cat. 13684S, Opal 620), CD11c (clone 5D11, Sigma Cell Marque cat. 111M-15, Opal 650), CD68 (clone KP1, Dako cat. M0814, Opal 690), and pan-CK (clone AE1/AE3, Dako cat. M351501-2, Opal 540) on a Bond RX autostainer (Leica Biosystems). Slides were dewaxed (Leica), heat treated in ER2 or ER1 antigen retrieval buffer depending on the antibody for 20 min at 93 °C (Leica), blocked in Antibody (Ab) Diluent (Akoya Biosciences), incubated for 30 min with the primary Ab, 10 min with horseradish peroxidase (HRP)-conjugated secondary polymer (anti-rabbit and anti-mouse, Akoya Biosciences), and 10 min with HRP-reactive OPAL fluorescent reagents (Akoya Biosciences). Slides were washed between staining steps with Bond Wash (Leica) and stripped between each round of staining with heat treatment in antigen retrieval buffer. After the final heat treatment in antigen retrieval buffer, the slides were stained with spectral DAPI (Akoya Biosciences), and coverslipped with Prolong Diamond mounting media (Thermo Fisher).

Strait A.A., Woolaver R.A., Hall S.C., Young C.D., Karam S.D., Jimeno A., Lan Y., Raben D., Wang J.H, & Wang X.J. (2021). Distinct immune microenvironment profiles of therapeutic responders emerge in combined TGFβ/PD-L1 blockade-treated squamous cell carcinoma. Communications Biology, 4, 1005.

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Multiplex Immunohistochemistry for Synovial Complement

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After confirming the integrity and architecture of the synovial biopsies, simultaneous staining for the seven CS proteins as above was performed. To detect and quantitate levels of seven complement proteins and inhibitors using MIHC, Early RA synovial sections after dewaxing (Leica) were heat treated in epitope retrieval solution 2 (ER2, EDTA buffer pH 8.9–9.1) or epitope retrieval solution 1 (ER1, citrate buffer pH 5.9–6.1) depending on the antibody for 20 min at 93 °C (Leica), blocked in antibody (Ab) Diluent (Akoya Biosciences®, Marlborough, MA), incubated for 30 min with the primary Ab, 10 min with horseradish peroxidase (HRP)-conjugated secondary polymer (anti-rabbit and anti-mouse, Akoya Biosciences), and 10 min with HRP-reactive OPAL fluorescent reagents (Akoya Biosciences). Slides were washed between staining steps with Bond Wash (Leica) and stripped between each round of staining with heat treatment in antigen retrieval buffer. After the final heat treatment in antigen retrieval buffer, the slides were stained with spectral DAPI (Akoya Biosciences), and coverslipped with Prolong Diamond mounting media (Thermo Fisher). To detect cell nuclei in the synovium, 4′, 6-diamidino-2-phenylindole (DAPI) was used.

Banda N.K., Deane K.D., Bemis E.A., Strickland C., Seifert J., Jordan K., Goldman K., Morgan B.P., Moreland L.W., Lewis M.J., Pitzalis C, & Holers V.M. (2022). Analysis of Complement Gene Expression, Clinical Associations, and Biodistribution of Complement Proteins in the Synovium of Early Rheumatoid Arthritis Patients Reveals Unique Pathophysiologic Features. Journal of immunology (Baltimore, Md. : 1950), 208(11), 2482-2496.

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Multiplex Immunohistochemistry for Head and Neck Cancer

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Seven-colour multiplex immunohistochemistry was performed with the OPAL Polaris system (Akoya Biosciences). Four-to-five-micrometre sections of formalin-fixed paraffin-embedded tumours from the patients with HNSCC were deparaffinized, hydrated and stained manually with anti-CD3 (clone F7.2.38, Dako), CD8 (clone C8/144B, eBiosciences), CD20 (clone L26, Invitrogen), TCF-1 (clone C63D9, Cell Signaling Technologies (CST)), PD-1 (clone D4W2J, CST) and cytokeratin (clone AE1/3, Dako) antibodies. Heat induced epitope retrieval (HIER) in EDTA (pH 9) or citrate (pH 6) buffer was performed before blocking non-specific binding and staining the tissues with the primary antibodies. The sections were sequentially stained with each primary, HRP-conjugated secondary antibody, tyramide signal amplification, and OPAL fluorophore according to the manufacturer’s instructions. OPAL 480, 520, 570, 620, 690 and 780 dyes were used. The sections were counterstained with spectral DAPI (Akoya Biosciences). The stained slides were imaged and scanned using the Vectra Polaris multispectral imaging system.

Eberhardt C.S., Kissick H.T., Patel M.R., Cardenas M.A., Prokhnevska N., Obeng R.C., Nasti T.H., Griffith C.C., Im S.J., Wang X., Shin D.M., Carrington M., Chen Z.G., Sidney J., Sette A., Saba N.F., Wieland A, & Ahmed R. (2021). Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer. Nature, 597(7875), 279-284.

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Multiplex Immunohistochemistry with Antigen Retrieval

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Antigen retrieval and antibody stripping steps were performed at 100°C with all other steps at ambient temperature. Endogenous peroxidase was blocked with 3% H2O2 for 8 minutes followed by protein blocking with TCT buffer (0.05M Tris, 0.15M NaCl, 0.25% Casein, 0.1% Tween 20, pH 7.6 +/− 0.1) for 30 minutes. The first primary antibody (position 1) was applied for 60 minutes followed by the secondary antibody application for 10 minutes and the application of the tertiary TSA-amplification reagent (OPAL fluor, Akoya Biosciences, Menlo Park, CA) for 10 minutes. The primary and secondary antibodies were stripped with retrieval solution for 20 minutes before repeating the process with the second primary antibody (position 2) starting with a new application of 3% H2O2. The process was repeated until all positions were completed; however, there was no stripping step after the last position. Slides were removed from the autostainer and stained with Spectral DAPI (Akoya) for 5 minutes, rinsed for 5 minutes, and cover-slipped with Prolong Gold Antifade reagent (Invitrogen/Life Technologies, Grand Island, NY).

Petit M.M., Fradelizi J., Golsteyn R.M., Ayoubi T.A., Menichi B., Louvard D., Van de Ven W.J, & Friederich E. (2000). LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity. Molecular Biology of the Cell, 11(1), 117-129.

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