Opal mIF staining was performed by the UCLA Translational Pathology Core Laboratory based on tyramide signal amplification (TSA). The Opal Polaris 7-Color Automation IHC Kit (Cat#NEL871001KT, Akoya Biosciences, Marlborough, MA, USA) was used with ER2 30 min retrieval and 1 h room temperature incubation. Staining was performed consecutively using each of the following antibodies: Paired Box 8 (PAX8, prediluted rabbit polyclonal antibody 363A-18, Cell Marque, Rocklin, CA, USA), calcyphosine (CAPS, Atlas Antibodies Cat#HPA043520, RRID:AB 10964138, 1:1000 dilution, Sigma-Aldrich, St. Louis, MO, USA), and tubulin beta 4 (TUBB4, Sigma-Aldrich, St. Louis, MO, USA), monoclonal anti-acetylated tubulin T7451, 1:500 dilution), and 4′,6-diamidino-2-phenylindole (DAPI) (Cat# SKU FP1490, Akoya Biosciences, Marlborough, MA, USA). The slides were scanned at 20X with the Vectra Polaris scanner (Akoya Biosciences, Marlborough, MA, USA) and data from the multispectral camera were analyzed using the imaging InForm automated image analysis software (Akoya Biosciences, Marlborough, MA, USA). Images were scored at 10X magnification using Phenochart whole slide viewer (Akoya Biosciences, Marlborough, MA, USA).
Opal polaris 7 color automation ihc kit
Manufactured by Akoya Biosciences
Sourced in United States
The Opal Polaris 7-Color Automation IHC Kit is a laboratory equipment product designed for automated immunohistochemistry (IHC) staining. It provides a 7-color multiplex IHC solution for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Bond rx system, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (2 mentions)
Phenochart whole slide viewer, by Akoya Biosciences (1 mentions)
Tubb4, by Merck Group (1 mentions)
Vectra polaris scanner, by Akoya Biosciences (1 mentions)
Inform automated image analysis software, by Akoya Biosciences (1 mentions)
Ab75813, by Abcam (1 mentions)
Ab178089, by Abcam (1 mentions)
Ab20034, by Abcam (1 mentions)
Vectra polaris quantitative pathology imaging system, by Akoya Biosciences (1 mentions)
A0452, by Abcam (1 mentions)
Vectra polaris, by Akoya Biosciences (1 mentions)
Bond rx, by Leica (1 mentions)
Panck, by Agilent Technologies (1 mentions)
4 protocols using opal polaris 7 color automation ihc kit
1
Opal Multicolor Immunofluorescence Staining
2
Quantifying CCL5 and PD-L1 in Tumor Samples
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IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described 26 . MIF staining was conducted using the Akoya OPAL Polaris 7-Color Automation IHC kit (NEL871001KT). FFPE tissue slides were first deparaffinized in a BOND RX system (Leica Biosystems), followed by sequential incubation with primary antibodies. After this, the samples were incubated with secondary antibodies and corresponding reactive Opal fluorophores. Nucleic acids were stained with DAPI. The quantities of various cell populations were expressed as the number of stained cells per square millimeter and as the percentage of positively stained cells in all nucleated cells. Opal immunofluorescence (mIF) was implemented to determine the presence of tertiary lymphoid structures (TLS), an abundance of tumor-associated lymphocytes (TILs) and other cells, and PD-L1 expression in relation to CCL5 expression on a multispectral imaging system (Vectra® Polaris™, Shanghai, China).
3
Multiplex IHC Analysis of Tumor Microenvironment
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Thirty FFPE samples were subjected to assessment of mIHC using the Akoya OPAL Polaris 7-Color Automation IHC kit (NEL871001KT). FFPE tissue samples were deparaffinized in a BOND RX system (Leica Biosystems) and then incubated sequentially with primary antibodies targeting CD68 (Abcam, ab213363, 1:1000), PD-L1 (CST, E1L3N, 13684S, 1:400), PD-1 (CST, D4W2J, 86163S, 1:200), CD163 (Abcam, ab182422, 1:500), CD3 (Dako, A0452), CD8 (Abcam, ab178089, 1:100), CD4 (Abcam, ab133616, 1:100), CD20 (Dako, L26, IR604), CD56 (Abcam, ab75813, 1:100), FOXP3 (Abcam, ab20034, 1:100) and pan-CK (Abcam, ab7753, 1:100) (Akoya Biosciences). Next, secondary antibodies and reactive Opal fluorophores were incubated. DAPI was used to stain the nucleic acids. Multiplex stained slides were scanned at 20 nm wavelength intervals from 440 nm to 780 nm with a fixed exposure time and an absolute magnification of 200 utilizing an Akoya Biosciences Vectra Polaris Quantitative Pathology Imaging System. All scans for each slide were then superimposed to create a single image. Multilayer images were imported into APTIME software (3D Medicines Inc.) for quantitative image analysis. Pan-CK staining was used to distinguish the tumor parenchyma and stroma. The numbers of stained cells per square millimeter in all nucleated cells were used to express the amounts of distinct cell types.
4
Multiplexed Immunofluorescence Tumor Profiling
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Tumor-immune spatial heterogeneity was quantified through histologic analysis. Multiplex immunostaining was performed using an Opal Polaris 7-Color Automation IHC Kit (Akoya Biosciences, Marlborough, MA; Cat#NEL871001KT), an optimized multiplex for CD8 (Cell Marque, Cat#108M-95), CD4 (Cell Marque, Cat#104R-25), FoxP3 (Cell Signaling, Cat#12653S), CD68 (Cell Marque, Cat#168M-95), CD20 (Cell Marque, Cat#120M-85), panCK (Dako/Agilent, Cat#M3515) and DAPI (Akoya, Cat#210713036). Analysis was conducted on a Leica Bond Rx fully automated autostainer (Leica Biosystems, Deer Park, IL). Biomarkers that could co-localize in the same cellular compartment were paired with a spectrally separated Opal fluorophore to avoid potential spectral interference, as recommended by the manufacturer. All multiplex immunofluorescence slides were scanned on an Akoya Vectra Polaris (Akoya Biosciences, Marlborough, MA) at 20X using MOTiF™ protocol, which generates a single unmixed whole slide scan of up to 7 colors. This single image facilitates a rapid application of digital image analysis across the entire slide in a streamlined workflow, without the requirement of stitching many spectrally unmixed image tiles. Thereafter, whole slide images were loaded into InForm image analysis software for automated cell type density quantification.
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