Mice were sacrificed at 6 hours, day 1, day 2, day 3, and day 7 after the virus challenge for collection of lung tissues. Lung tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. Five-micrometer sections were cut and stained with H&E. The pathology scores were calculated following a methodology we previously published (He et al., 2020 (link)). For immunohistochemical (IHC) assays, paraffin sections of the lung were de-waxed and then subjected to heat treatment in citrate buffer, followed by quenching of endogenous peroxidase activity using 0.3% H2O2 in methanol. Sections were blocked for 1 hour (hr) with Fc Receptor Blocker and then incubated overnight at 4°C with F4/80 antibody (CST No.70076, 1:500). Antibody binding was detected using ZSGB System reagents (ZSGB, Beijing). After counterstaining with haematoxylin, the slides were incubated with CD4-specific antibody (Abcam No. ab183685, 1:1000) and CD8-specific antibody (LSBio No.LS-C43572, 1:200), and multiplexed immunofluorescence staining was then performed using Opal 7-color Manual IHC Kit (Akoya,USA). The stained slides were scanned by TissueFAXS 200 (TissueGnostics). The acquired images were analyzed by Strata Quest software to assess the number of F4/80+, CD4+, CD8+ cell and the inflammatory infiltration areas.
Opal 7 color manual ihc kit
Manufactured by Akoya Biosciences
Sourced in United States
The Opal 7-Color Manual IHC Kit is a laboratory equipment product designed for multiplex immunohistochemistry (IHC) analysis. It enables the simultaneous detection and visualization of up to 7 protein targets on a single tissue section using a manual staining workflow.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (4 mentions)
Tissuefaxs 200, by TissueGnostics (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Vectra 3, by Akoya Biosciences (1 mentions)
Z2 microscope, by Zeiss (1 mentions)
Spectral dapi, by Akoya Biosciences (1 mentions)
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Metasystem automated slide scanner, by MetaSystems (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
F4 80 antibody, by Cell Signaling Technology (1 mentions)
Inform software v2, by Akoya Biosciences (1 mentions)
Anti ass1, by Thermo Fisher Scientific (1 mentions)
Anti collagen type 3, by Abcam (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Superfrost plus microscope slides, by Avantor (1 mentions)
Zb 2301, by ZSGB-BIO (1 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions)
Cd8 clone c8 144b, by Agilent Technologies (1 mentions)
Pd 1 clone eh33, by Cell Signaling Technology (1 mentions)
Cd103, by Abcam (1 mentions)
21 protocols using opal 7 color manual ihc kit
1
Lung Pathology Analysis in Virus-Challenged Mice
2
Multiparametric Immunohistochemistry of Tumor Tissues
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Tissues with paraformaldehyde-fixed and paraffin-embedded were cut into 2 µm sections. After deparaffinized and rehydrated, the slices were fixed by 4% paraformaldehyde for 20 min and then antigen retrieval as normal IHC. mIHC staining was performed by using the Opal 7-Color Manual IHC Kit (NEL811001KT, Akoya Biosciences). The tumor sections were blocked in Opal Antibody Diluent/Block for 12 min at room temperature. Primary antibodies were incubated for 1 h at 37 °C or overnight at 4 °C. Following washing in TBST, the sections were incubated with secondary Abs Opal Polymer HRP Ms+Rb for 10 min at 37 °C. Sections were then washed in TBST and stained for 10 min with fluorescence staining diluted 1:100 in 1× Plus Amplification Diluent. All the slides were stained with DAPI for 5 min at room temperature and imaged by Akoya Vectra3. The images were sequentially spectrally unmixed by Akoya phenoptics inForm software.
3
Multiplexed Fluorescent IHC Protocol
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Fluorescent multiplexed IHC was performed with the Opal 7-color Manual IHC Kit (Akoya, NEL861001KT) according to the manufacturer’s protocol. In brief, slides were deparaffinized, microwave-treated in epitope retrieval buffer for 45 s at 100% power and an additional 15 min at 20% power, blocked in Opal Antibody Diluent/Block at room temperature for 10 min, and incubated with the specific primary antibody overnight at 4°C, 10 min with the secondary horseradish peroxidase (HRP)–conjugated antibody Polymer HRP Ms + Rb at room temperature, and 10 min with Opal fluorophore working solution. Slides were rinsed between staining steps with 1× tris-buffered saline with Tween 20 buffer and stripped between each round of staining via microwave treatment in antigen retrieval buffer. After the final microwave treatment, slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min followed by mounting. Images were acquired with a confocal microscope (LSM 880, Zeiss). Table S8 lists the antibodies used.
4
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].
5
Multiplex Immunohistochemistry for Tumor Microenvironment Analysis
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Slides were prepared from FFPE specimens of mouse tumor tissues. Multiplex IHC consecutive staining on a single slide was performed with use of the Opal 7-Color Manual IHC Kit (AKOYA Biosciences, #NEL811001KT) to analyze the TIME. A series of sequential cycles of staining, image scanning, and destaining of chromogenic substrate was performed on FFPE tissue samples. Visualization of 7-Color Opal slides was performed by using a Mantra or Vectra quantitative pathology imaging system. Each system uses multispectral imaging for quantitative unmixing of many fluorophores and tissue autofluorescence. The antibodies used for multiplex IHC are described in the Supplementary Materials and Methods section.
6
Quantifying Tumor-Associated Macrophages
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Mice were sacrificed on day 15 after tumor inoculation. The entire tumors were fixed with 4% paraformaldehyde and then embedded in paraffin. Sections of 5 µm in size were cut from the embedding tissue. The sections were further deparaffinized and rehydrated through xylene and graded ethanol series, and subjected to heat treatment in citrate buffer, followed by quenching of endogenous peroxidase activity using 0.3% H2O2 in methanol. Sections were blocked for 1 h with Fc Receptor Blocker and incubated separately with CD206 antibody (ab300621, 1:1000; Abcam) and F4/80 antibody (70076, 1:500; Cell Signaling Technology). Multiplexed immunofluorescence staining was then performed using Opal 7-color Manual IHC Kit (Akoya, USA). After washing, the stained slides were stained with DAPI for 2 min and scanned by TissueFAXS 200 (TissueGnostics). The acquired images were analyzed by Strata Quest software to assess the number of F4/80+ and CD206+.
7
Multiplex IHC with Opal Fluorophores
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The Opal 7-Color Manual IHC Kit (AKOYA Biosciences, NEL811001KT) was used for multiplex IHC consecutive staining on a single slide. A series of sequential cycles of blocking, epitope retrieval, primary and secondary Ab incubation, and opal fluorophore staining were performed on FFPE tissue samples. We visualized 3-color Opal slides through Mantra or Vectra Quantitative Pathology Imaging Systems. The systems use multispectral imaging for quantitative unmixing of fluorophores and tissue autofluorescence. The primary Abs used for multiplex IHC were anti-CD3ε (1:100; catalog 78588, Cell Signaling Technology) and anti–PD-1 (1:150; catalog 84651, Cell Signaling Technology).
8
Multiplex Immunofluorescence Analysis of CDKN2A, CD4, and CD8
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Briefly, the slides were dewaxed and rehydrated, and antigen retrieval was performed. The endogenous peroxidase and Fc receptor were blocked as described in the IHC protocols. The samples were stained with rabbit monoclonal anti-CDKN2A (1:1,000. Abcam), rabbit monoclonal anti-CD4 (1:5, SP8, MXB), and rabbit monoclonal anti-CD8 (1:5, SP16, MXB). The Opal 7-Color Manual IHC Kit (Akoya Biosciences, DE, USA) was used to generate immunofluorescent signals, using TSA dye 520 (1:100, anti-CDKN2A), dye 570 (1:100, anti-hCD8), and dye 620 (1:100, anti-hCD4). Spectral 4,6-diamidino-2-phenylindole (DAPI) (1:10, Akoya) was used to counterstain the samples. The Vectra® 3.0 Automated Quantitative Pathology Imaging System was used for multi-slide imaging. The image data were extracted using the inForm® Software v2.6 (Akoya).
9
Multiparametric Tissue Immunohistochemistry
10
Immunohistochemical Analysis of FFPE Tumor Tissues
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From each FFPE tumor tissue block, 2 μm sections were prepared on Superfrost® Plus microscope slides (VWR, Leuven, Belgium) and confirmed by a pathologist for the presence of tumor tissue. The slides were deparaffinized at 60 °C for two hours, washed in xylene for 3 × 10 min, hydrated with descending grades of ethanol (100%, 96%, and 70%; Penta, Prague, Czech Republic), and fixed for 20 min in neutral buffered formalin, pH 7.2–7.4 (Diapath, Martinengo, Italy). Heat-induced epitope retrieval (HIER) was carried out by using microwave (96–98 °C) in buffer of pH 9.0 (AR9, Tris-EDTA; Zytomed Systems, Berlin, Germany) or pH 6.0 (AR6, citrate buffer; Akoya Biosciences, Menlo Park, CA, USA). After 10 min of blocking using Antibody Diluent/Block (Akoya Biosciences), the slides were incubated with the primary antibodies listed in Table 1 and subsequently stained with the Opal™ 7-Color Manual IHC Kit (Akoya Biosciences), according to the manufacturers’ instructions. Validation of the antibodies was carried out on samples of non-malignant tonsillar and HNSCC tissue. To verify staining specificity, the corresponding isotype controls, as well as no-primary controls, were performed using the appropriate isotype antibody or Antibody Diluent/Block, respectively.
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