Opal620

Manufactured by PerkinElmer

The Opal620 is a compact and versatile benchtop flow cytometer designed for multiplexed tissue analysis. It utilizes up to 7 fluorescence channels and provides high-speed data acquisition for simultaneous detection of multiple cellular markers. The Opal620 is capable of analyzing a variety of sample types, including cells, tissues, and 3D models.

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Lab products found in correlation

Opal 520, by PerkinElmer (3 mentions) Opal 570, by PerkinElmer (2 mentions) Opal 690, by PerkinElmer (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by PerkinElmer (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Mantra quantitative pathology workstation, by PerkinElmer (1 mentions) Opal 650, by PerkinElmer (1 mentions) Inform advanced image analysis software, by PerkinElmer (1 mentions) Opal 7 color ihc kit, by PerkinElmer (1 mentions) Scn400 whole slide scanner, by Leica (1 mentions) Fluoromount g, by Thermo Fisher Scientific (1 mentions) Vectra imaging software, by PerkinElmer (1 mentions) Ab272504, by Abcam (1 mentions) Syto13, by Thermo Fisher Scientific (1 mentions) Bond rx, by Leica (1 mentions) Axioscanner, by Zeiss (1 mentions)

5 protocols using opal620

Multiplex Immunohistochemistry for Tumor Microenvironment

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mIHC was performed using the Opal 7-color IHC kit (PerkinElmer) as previously described.25 (link) Briefly, FFPE sections (4 µm thick) were dewaxed and rehydrated. Antigen retrieval was performed in citrate buffer (PH=6.0), and endogenous peroxidase was quenched using 3% H₂O₂ for 15 min. The slides were blocked with 2% bovine serum albumin for 15 min and then incubated with primary antibodies of two panels overnight, including CD3, CD4, CD20, Foxp3 and PD-1; CD8, CD56, CD68, CD163 and PD-L1 (online supplemental table 1). Then, slides were incubated with corresponding HRP-conjugated secondary antibodies and fluorescent dyes with the following order: Opal540, Opal570, Opal620, Opal650 and Opal690 (PerkinElmer). Nuclei was stained with DAPI (PerkinElmer). Subsequently, the slides were scanned and imaged using the Mantra Quantitative Pathology Workstation (PerkinElmer, Waltham, Massachusetts, USA). Images were obtained for the following analysis using the inForm Advanced Image Analysis software (V.2.4.2, PerkinElmer). Multispectral images were unmixed using spectral libraries obtained from images of single-stained slides for each marker. For subregions with multiple TLSs, the densities and frequencies of each marker were averaged to represent the cellular composition.

Zhang C., Wang X.Y., Zuo J.L., Wang X.F., Feng X.W., Zhang B., Li Y.T., Yi C.H., Zhang P., Ma X.C., Chen Z.M., Ma Y., Han J.H., Tao B.R., Zhang R., Wang T.Q., Tong L., Gu W., Wang S.Y., Zheng X.F., Yuan W.K., Kan Z.J., Fan J., Hu X.Y., Li J., Zhang C, & Chen J.H. (2023). Localization and density of tertiary lymphoid structures associate with molecular subtype and clinical outcome in colorectal cancer liver metastases. Journal for Immunotherapy of Cancer, 11(2), e006425.

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Immunohistochemical Analysis of Tumor Markers

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The tumor tissue was initially fixed with 4% paraformaldehyde, embedded in paraffin and cut into 5 μm thick sections. The sections were blocked with 10% goat serum and incubated with anti-TOP2A (1:200, Cat#12286, CST) or anti-CENPF (1:200, Cat#ab224813, Abcam), or anti-ki-67 (1:200, M00254-9, Boster) antibodies at 4°Cat night. After washing with PBS, the sections were incubated with a secondary antibody conjugated with anti-mouse or rabbit horseradish peroxidase at room temperature for 1 hour. Paraffin sections were stained with DAB and hematoxylin. The IHC section slides were observed by a microscope (Nikon, Japan). Multiplexed immunofluorescence was performed as we previously described.⁸¹ (link) Opal-520, Opal-620 (PerkinElmer) and 4′,6-diamidino-2-phenylindole (DAPI) were applied to each antibody for visualization.

Zhao H.C., Chen C.Z., Tian Y.Z., Song H.Q., Wang X.X., Li Y.J., He J.F, & Zhao H.L. (2023). CD168+ macrophages promote hepatocellular carcinoma tumor stemness and progression through TOP2A/β-catenin/YAP1 axis. iScience, 26(6), 106862.

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Paraffin Embedding and Immunofluorescence Staining

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Tumors were fixed in Z7 zinc based fixative[31 (link)] overnight. Tissue was then processed for paraffin tissue sections. Tissue was dehydrated through graded alcohol to xylene, incubated in molten paraffin using a Tissue-Tek automated tissue processor (Sakura, Torrance, CA), and then embedded in paraffin. 5 μm sections were cut and mounted for analysis. Trichrome stain was performed according to manufacturer’s protocol (Cardinal Health, Dublin, OH). Primary antibody binding was visualized with Alexa Fluor 488 (Thermo Fisher Scientific), Opal 520, or Opal 620 secondary antibodies (Perkin Elmer, Boston, MA) following either ImmPRESS HRP anti-rabbit or anti-rat polymer incubation (Vector laboratories). Sections were stained with DAPI (Perkin Elmer) for nuclear staining and mounted with FluoromountG (Thermo Fisher Scientific). Images were acquired using: Vectra imaging software (Perkin Elmer); a Leica SCN400 whole slide scanner or a SCN400F fluorescence whole slide scanner. All images displayed in the manuscript are representative of the entire tumor and their respective experimental cohort. NIH image J software was used to separate into their single marker components, to set threshold to minimize back ground noise, and to quantify the positive pixel area in each image. Minimum of 4 tumors per cohort were utilized for analysis.

Gunderson A.J., Yamazaki T., McCarty K., Phillips M., Alice A., Bambina S., Zebertavage L., Friedman D., Cottam B., Newell P., Gough M.J., Crittenden M.R., Van der Veken P, & Young K.H. (2019). Blockade of fibroblast activation protein in combination with radiation treatment in murine models of pancreatic adenocarcinoma. PLoS ONE, 14(2), e0211117.

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Multiplex Immunohistochemistry for Tumor-Infiltrating Lymphocytes

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Paraffin-embedded tumor sections were deparaffinized, blocked, and stained with primary antibodies as described^{21 (link)}. Sequential staining was achieved with anti-mouse CD4 (clone 4SM95), Opal520 (PerkinElmer), CD8 (4SM15), and Opal620 with washes performed in Amplification Plus buffer (PerkinElmer). Nuclei counterstaining was achieved with Spectral DAPI. Slides were imaged on a Vectra Polaris Imager (PerkinElmer) and analyzed with QuPath software^{22 (link)}.

Lee M.Y., Metenou S., Brough D.E., Sabzevari H., Bai K., Jochems C., Schlom J, & Allen C.T. (2021). Preclinical study of a novel therapeutic vaccine for recurrent respiratory papillomatosis. NPJ Vaccines, 6, 86.

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SARS-CoV-2 Spike Protein Immunohistochemistry and RNA Detection

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Immunohistochemistry was performed on a Leica Bond-RX autostainer (Leica Biosystems, USA) with antibody targeting SARS-CoV-2 spike protein (Abcam, ab272504) at 2 μg·mL⁻¹. Heat-induced epitope retrieval was performed in buffer ER1 at 100°C for 20 min, and the signal visualised with 3,3′-diaminobenzidine substrate. Slides were imaged using a Zeiss Axioscanner (Carl Zeiss, Germany) and immunohistochemistry was scored by a pathologist for bronchiolar epithelium, type 2 pneumocytes, interstitial lymphocytes and alveolar macrophage compartments.
RNAscope probes (ACDBio, USA) targeting SARS-CoV-2 spike mRNA (nCoV2019, #848561-C3), angiotensin-converting enzyme (ACE)2 host receptor mRNA (#848151-C2) and host serine transmembrane protease, serine 2 (TMPRSS2) mRNA (#470341-C1) were used per manufacturer's instructions for automation on Leica Bond RX. DNA was visualised with Syto13 (ThermoFisher Scientific), channel 1 with Opal 570 (1:500), channel 2 with Opal 620 (1:1500) and channel 3 with Opal 690 (1:1500) (PerkinElmer). Fluorescent images were acquired with NanoString Mars prototype DSP at 20×.

Kulasinghe A., Tan C.W., Ribeiro dos Santos Miggiolaro A.F., Monkman J., SadeghiRad H., Bhuva D.D., Motta Junior J.D., Busatta Vaz de Paula C., Nagashima S., Baena C.P., Souza-Fonseca-Guimaraes P., de Noronha L., McCulloch T., Rossi G.R., Cooper C., Tang B., Short K.R., Davis M.J., Souza-Fonseca-Guimaraes F., Belz G.T, & O'Byrne K. (2022). Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures. The European Respiratory Journal, 59(6), 2101881.

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