Multispectral fluorescence staining was performed using the Opal 6-Plex Detection kit (Akoya Biosciences) using FFPE tissue sections. Slides were baked at 60°C for 15 minutes and deparaffinized with the Leica Bond Dewax solution (Leica Biosystems), followed by heat-based antigen retrieval using Bond Epitope Retrieval Solution 1 (Leica Biosystems) for 30 minutes. Using the Leica Bond Rx Automated Stainer (Leica Biosystems), slides were incubated with primary antibodies followed by the appropriate secondary horseradish peroxidase–conjugated polymer. Incubation was next performed with a unique Opal dye permitting fluorophore covalent bonding to the horseradish polymer. Heat-based retrieval with Bond Epitope Retrieval 1 (Leica Biosystems) was finally performed for 20 minutes. Slides were subjected to sequential rounds of staining. Primary antibodies, concentrations, and associated fluorophores are detailed in Supplemental Table 9 . Sections were counterstained with Spectral DAPI and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using the Vectra3 microscope (Akoya Biosciences) and Phenochart Whole Slide Viewer (Akoya Biosciences). Postacquisition image adjustments were performed using InForm Automated Image Analysis Software (Akoya Biosciences) and Fiji (74 (link)).
Opal 6 plex detection kit
Manufactured by Akoya Biosciences
Sourced in United States
The Opal 6-Plex Detection kit is a laboratory product designed for the simultaneous detection of up to six biomarkers in a single tissue sample. It is a multiplex immunofluorescence solution developed by Akoya Biosciences for use with their proprietary imaging technology.
Automatically generated - may contain errors
Lab products found in correlation
Bond dewax solution, by Leica (3 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (3 mentions)
Bond epitope retrieval solution 1, by Leica (3 mentions)
Vectra3 microscope, by Akoya Biosciences (3 mentions)
Phenochart whole slide viewer, by Akoya Biosciences (3 mentions)
Inform automated image analysis software, by Akoya Biosciences (3 mentions)
Bond epitope retrieval 1, by Leica (3 mentions)
Bond rx, by Leica (3 mentions)
Bond rx automated stainer, by Leica (2 mentions)
Inform software v2, by Akoya Biosciences (1 mentions)
Phenoimager ht automated quantitative pathology imaging system, by Akoya Biosciences (1 mentions)
Pan keratin, by Abcam (1 mentions)
Phenoimager ht system, by Akoya Biosciences (1 mentions)
Cd4 clone ep204, by Cell Marque (1 mentions)
Cytokeratin clone ae1 ae3, by Thermo Fisher Scientific (1 mentions)
Cd8 clone c8 144b, by Agilent Technologies (1 mentions)
Cd68 clone pg m1, by Agilent Technologies (1 mentions)
Mantra 2 quantitative pathology workstation, by Akoya Biosciences (1 mentions)
6 protocols using opal 6 plex detection kit
1
Multispectral Fluorescence Staining Protocol
2
Multiplex Fluorescence Staining for Tissue Analysis
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Multispectral fluorescence staining was performed using the Opal 6-Plex Detection kit (Akoya Biosciences, Marlborough, MA) using formalin-fixed, paraffin embedded tissue sections. Slides were baked at 60 °C for 15 min and deparaffinized with the Leica Bond Dewax solution (Leica Biosystems, Deer Park, IL), followed by heat-based antigen retrieval using Bond Epitope Retrieval Solution 1 (Leica Biosystems) for 30 min. Using the Leica Bond Rx™ Automated Stainer (Leica Biosystems), slides were incubated with primary antibodies followed by the appropriate secondary horseradish peroxidase-conjugated polymer. Incubation was next performed with a unique Opal dye permitting fluorophore covalent bonding to the horseradish polymer. Heat-based retrieval with Bond Epitope Retrieval 1 (Leica Biosystems) was finally performed for 20 min. Slides were subjected to sequential rounds of staining. Primary antibodies, concentrations, and associated fluorophores are detailed in Supplemental Table 9 . Sections were counterstained with Spectral DAPI and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using the Vectra3 microscope (Akoya Biosciences) and Phenochart Whole Slide Viewer (Akoya Biosciences). Post-acquisition image adjustments were performed using InForm Automated Image Analysis Software (Akoya Biosciences) and Fiji (76 (link)).
3
Multispectral Immunofluorescent Staining of FFPE Tissue
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The multispectral immunofluorescent (mIF) staining on formalin-fixed paraffin-embedded (FFPE) tissue sections were performed using the Opal 6-Plex Detection Kit (AKOYA Biosciences, catalog no. NEL821001KT) as described in our previous study (29 (link)). The mIF panel consisted of the following antibodies: CD8 (EPR20305, Abcam, 570), Pan Keratin (Wide Spectrum cytokeratin, Abcam, 480), and DAPI. Slides were imaged on the PhenoImager HT Automated Quantitative Pathology Imaging System (AKOYA Biosciences). Further analysis of the slides was performed using inForm Software v2.6.0 (AKOYA Biosciences).
4
Multiplex Immunofluorescence Staining of Biopsy Wounds
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Multiplex immunofluorescence staining was performed with Opal 6-Plex Detection Kit (NEL871001KT; Akoya Biosciences, Marlborough, MA) using 6 Opal dyes along with Spectral DAPI counterstain. FFPE tissue samples were prepared for staining by baking at 60°C for 1 h followed by three 10-min washes in xylene to remove paraffin. Samples were rehydrated via an ethanol gradient into deionized water. A six-step cycle of heat-induced epitope retrieval, blocking, primary antibody incubation, Polymer HRP mouse + rabbit secondary antibody incubation, Opal dye-conjugated tyramide deposition, and antibody stripping was performed for each of the six tissue biomarkers using a Leica BOND RX fully automated research stainer (Leica Biosystems). Slides were mounted with coverslips using ProlongGold and multispectral detection and imaging was performed using the PhenoImager HT System (Akoya). Analysis of the data was performed using InForm cell analysis software. A core biopsy wound is defined as a space or tract surrounded by fibrotic and/or cystic changes with inflammatory changes that follows the shape of the biopsy trocar used in the initial diagnostic biopsy. Adjacent and distant were defined as areas within 400 μm and >3 mm away from the border of biopsy wound, respectively.
5
Multiplex Immunofluorescence of Renal Cell Carcinoma
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In-House RCC samples (n = 4) after surgery were fixed in 4% paraformaldehyde, embedded in paraffin and five-micrometer sections were used for the immunofluorescence staining. Multiplex IHC was performed using Opal 6-plex Detection Kit (cat: NEL821001KT, Akoya Biosciences, Menlo Park, USA). A multiplex panel of immune markers was developed with antibodies against CXCR3 (clone EPR25373-32, cat: ab288437, dilution 1:200, Abcam, Cambridge, MA, USA), CD4 (clone EP204, cat: 104R-26, dilution 1:50, Cell Marque), CD8 (clone C8/144B, cat: M710301-2, dilution 1:200, Dako/Agilent, Santa Clara, CA, USA), CD68 (clone PG-M1, cat: M087601-2, dilution 1:250, Dako/Agilent), cytokeratin (clone AE1/AE3, cat: MA5-13156, dilution 1:500, Thermo-Fisher). The staining procedure was performed using an automated staining system (BOND-RX; Leica Biosystems, Vienna, Austria). To visualize cell nuclei, the tissue was stained with 4’,6-diamidino-2-phenylindole (spectral DAPI, Akoya Biosciences). Slides were scanned at 20x magnification using Mantra 2 Quantitative Pathology Workstation (Akoya Biosciences) and representative images from each tissue were acquired with the Mantra Snap software version 1.0.4. Image spectral deconvolution, multispectral image analysis and cell phenotyping was carried out using the InForm Tissue Analysis Software version 2.4.10 (Akoya Biosciences).
6
Multispectral Fluorescence Staining Protocol
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Multispectral fluorescence staining was performed using the Opal 6-Plex Detection kit (Akoya Biosciences) using FFPE tissue sections. Slides were baked at 60°C for 15 minutes and deparaffinized with the Leica Bond Dewax solution (Leica Biosystems), followed by heat-based antigen retrieval using Bond Epitope Retrieval Solution 1 (Leica Biosystems) for 30 minutes. Using the Leica Bond Rx Automated Stainer (Leica Biosystems), slides were incubated with primary antibodies followed by the appropriate secondary horseradish peroxidase–conjugated polymer. Incubation was next performed with a unique Opal dye permitting fluorophore covalent bonding to the horseradish polymer. Heat-based retrieval with Bond Epitope Retrieval 1 (Leica Biosystems) was finally performed for 20 minutes. Slides were subjected to sequential rounds of staining. Primary antibodies, concentrations, and associated fluorophores are detailed in Supplemental Table 9 . Sections were counterstained with Spectral DAPI and mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using the Vectra3 microscope (Akoya Biosciences) and Phenochart Whole Slide Viewer (Akoya Biosciences). Postacquisition image adjustments were performed using InForm Automated Image Analysis Software (Akoya Biosciences) and Fiji (74 (link)).
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