Paraffin-embedded sections of mouse hind paw tissue were de-waxed, followed by heat-induced antigen retrieval in EDTA (pH = 9.0) using a microwave oven. After blocking for 1 h with goat serum (ZhongShan JinQiao-Bio, China), the sections were incubated with primary antibodies targeting F4/80, CD206, and NOS2 (all from Abcam, London, UK) for 1hat room temperature, and then with an HRP-conjugated secondary antibody (Abcam). Detection was performed using the Opal Multiplexed Manual IHC Kit (PerkinElmer, USA). Finally, all the sections were counterstained with DAPI, sealed, and observed and recorded using a Vectra Polaris imaging system (PerkinElmer, USA). Staining was analyzed using inForm Cell Analysis software (PerkinElmer, USA).
Vectra polaris imaging system
Manufactured by PerkinElmer
Sourced in United States
The Vectra Polaris imaging system is a high-performance, automated digital pathology platform designed for advanced multiplex tissue analysis. It provides researchers with the ability to image and quantify multiple biomarkers simultaneously within a single tissue sample.
Automatically generated - may contain errors
Lab products found in correlation
Hardset mounting media, by Vector Laboratories (2 mentions)
Phenochart, by PerkinElmer (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Goat serum, by Abcam (1 mentions)
Inform cell analysis software, by PerkinElmer (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Dab 3 3 diaminobenzidine, by Vector Laboratories (1 mentions)
Vectastain elite abc avidin biotin complex kit, by Vector Laboratories (1 mentions)
Roti histokitt 2, by Carl Roth (1 mentions)
Axiocam hrc camera, by Zeiss (1 mentions)
Axioplan 2 imaging microscope, by Zeiss (1 mentions)
Inform software, by Akoya Biosciences (1 mentions)
H 1400, by Vector Laboratories (1 mentions)
Phenoptrreports, by Akoya Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (1 mentions)
Opal 520 reagent, by Akoya Biosciences (1 mentions)
Opal 570 reagent, by Akoya Biosciences (1 mentions)
Envision flex target retrieval solution of high ph, by Agilent Technologies (1 mentions)
Ab5694, by Abcam (1 mentions)
8 protocols using vectra polaris imaging system
1
Multiplex Immunohistochemistry Analysis
2
Immunohistochemical Staining of FFPE Tissues
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FFPE tissue was cut into 2 μm sections on a microtome and de-paraffinized. After antigen retrieval slides were incubated with primary antibody (the primary antibodies used are listed in Table S3 ) over night at 4 °C and washed with Tris-HCL (Tris hydrochloride) buffer (pH 7.5) followed by a secondary antibody. Antibodies were detected with the Vectastain Elite ABC (avidin-biotin complex) kit (Vector) using DAB (3,3′-diaminobenzidine) (Vector Laboratories and Dako) for brown stainings or AEC (3-Amino-9-ethylcarbazole) (Thermo Fisher Scientific) for magenta stainings. The slides were counterstained with hematoxylin (Vector Laboratories) and mounted with Roti®-Histokitt II (Carl Roth, Germany). Images were captured on an Axioplan2 imaging microscope (Carl Zeiss) equipped with an AxioCamHRc Camera (Carl Zeiss) or slides were scanned with a Vectra Polaris imaging system (PerkinElmer, Hopkinton, MA, USA) and quantified by ImageJ software.
3
Quantifying Immune Cell Populations
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Multispectral IF‐stained slides were imaged using the Vectra Polaris imaging system (Perkin Elmer, Waltham, MA, USA). Following initial whole slide scans, high‐power (40×x objective magnification) images were taken for analysis from representative sections (two from the tumour centre and one from the invasive margin). Tissue was segmented into epithelia and stroma by training on DAPI and cytokeratin stains and the following cell populations defined and quantified: total T‐cells (CD3+), cytotoxic T‐cells (CD3+CD8+), and T regulatory cells (CD3+FoxP3+).
4
Multiplex Immunofluorescent Staining of FFPE Tissues
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Multiplex immunofluorescent staining was performed using Opal Immunology Discovery kit (Akoya biosciences) on formalin fixed paraffin embedded tissues according to the manufacturer's instructions. Briefly, sections were deparaffinized using xylene and hydrated using gradient ethanol ending with a distilled water wash. Microwave treatment was performed in AR6 buffer for antigen retrieval. Then slides were incubated with a primary antibody followed by secondary antibody. Opal fluorophore solution was added to the slide and incubated for 10 min at room temperature. Microwave treatment was performed again to remove the first primary antibody. Steps were repeated for subsequent primary antibodies to achieve multiplex IF staining. After antibody staining, slides were incubated in DAPI solution for 5 min at room temperature, washed several times, and then mounted with coverslip. The IF signals were detected by Vectra Polaris Imaging System (PerkinElmer) and analyzed by inForm software (Akoya).
5
Multiplex Immunohistochemistry Staining Protocol
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Formalin-fixed and paraffin-embedded tissue sections (3μm) were de-paraffinized and rehydrated. Next, heat-induced epitope retrieval (HIER) was performed, followed by blocking with 3% hydrogen peroxide in TBST for 10 min and staining with the multiplex mIHC kit (PerkinElmer, NEL861001KT, Shanghai Kelin Institute). Briefly, after the first primary antibody staining, slides were incubated using the HRP-polymer detection system for 10 min, then visualization using Opal TSA working solution (1:100) for another 10 min. Afterward, antigen retrieval was conducted again to prepare the slides for the next antibody. Using this Opal staining method, primary antibodies were applied sequentially. Lastly, slides were counterstained with DAPI (Sigma, 1:1000) for nuclei visualization and subsequently coverslipped using the Hardset mounting media (VectaShield, H-1400).
All tissue sections that underwent multiplex fluorescent staining for each fluorophore were imaged using the Vectra Polaris imaging system (PerkinElmer, Shanghai Kelin Institute) under the appropriate fluorescent filters to produce the spectral library required for multispectral analysis. A whole slide scan of the multiplex tissue sections produced multispectral fluorescent images visualized in Phenochart (PerkinElmer) and imaging at 200× magnification.
All tissue sections that underwent multiplex fluorescent staining for each fluorophore were imaged using the Vectra Polaris imaging system (PerkinElmer, Shanghai Kelin Institute) under the appropriate fluorescent filters to produce the spectral library required for multispectral analysis. A whole slide scan of the multiplex tissue sections produced multispectral fluorescent images visualized in Phenochart (PerkinElmer) and imaging at 200× magnification.
6
Multiplex immunohistochemistry for tissue analysis
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Formalin-fixed and paraffin-embedded tissue Sects. (3 mm) were de-paraffinized and rehydrated. Next, heat-induced epitope retrieval (HIER) was performed, followed by blocking with 3% hydrogen peroxide in TBST for 10 min and staining with the multiplex mIHC kit (PerkinElmer, NEL861001KT, Shanghai Kelin Institute). Briefly, after the first primary antibody staining, slides were incubated using the HRP-polymer detection system for 10 min, then visualization using Opal TSA working solution (1:100) for another 10 min. Afterward, antigen retrieval was conducted again to prepare the slides for the next antibody. Using this Opal staining method, primary antibodies were applied sequentially. Lastly, slides were counterstained with DAPI (Sigma, 1:1000) for nuclei visualization and subsequently coverslipped using the Hardset mounting media (VectaShield, H-1400).
All tissue sections that underwent multiplex fluorescent staining for each fluorophore were imaged using the Vectra Polaris imaging system (PerkinElmer, Shanghai Kelin Institute) under the appropriate fluorescent filters to produce the spectral library required for multispectral analysis. A whole slide scan of the multiplex tissue sections produced multispectral fluorescent images visualized in Phenochart (PerkinElmer).
All tissue sections that underwent multiplex fluorescent staining for each fluorophore were imaged using the Vectra Polaris imaging system (PerkinElmer, Shanghai Kelin Institute) under the appropriate fluorescent filters to produce the spectral library required for multispectral analysis. A whole slide scan of the multiplex tissue sections produced multispectral fluorescent images visualized in Phenochart (PerkinElmer).
7
Multispectral Imaging of Immune Cells in Tumor Samples
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Methods for multispectral imaging have previously been published and the methods herein are described in brief33 (link),39 (link). Following the manufacturer’s protocol, FFPE slides were sequentially stained using Opal IHC Multiplex Assay (PerkinElmer, Waltham, MA) by the Human Immune Monitoring Shared Resource at the University of Colorado Anschutz Medical Campus. The Vectra Polaris Imaging System (PerkinElmer) scanned the whole slide. The panel used for the Vectra Polaris were as follows in sequential order: CD11b, CD64, EpCAM. CD11c, B220, CD8, F4/80. Approximately 5 regions of interest (ROIs) were evaluated per tumor. Images were analyzed using inForm Tissue Analysis Software (v2.4.8, Akoya, Menlo Park, CA) to un-mix fluorochromes, remove autofluorescence, segment tissue and cells, and phenotype cells. Data analysis was performed as previously described by our group21 (link),39 (link). Briefly, data acquired from inForm was analyzed using the R package Akoya Biosciences phenoptrReports. The count_phenotypes function was used to aggregate phenotype counts for each slide. Data are presented as cell counts.
8
Multiplexed Immunostaining of Paraffin Sections
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After dewaxing and rehydration of the paraffin-embedded sections, the antigen retrieval was conducted using Envision Flex Target Retrieval Solution of high pH (DAKO). For FSTL1 and aSMA staining, the sections were blocked with Opal Antibody Diluent/Block (AKOYA), followed by incubation with FSTL1 (1:100, Abcam, ab71548) for 1 hour at room temperature. Signal was detected by incubating Opal Polymer horseradish peroxidase (HRP), Mouse plus Rabbit (AKOYA) for 30 minutes, followed by Opal 570 Reagent (AKOYA). Another round of antigen retrieval and antibody stripping were conducted before incubating with aSMA (1:100, Abcam, ab5694) for 1 hour at room temperature. Signal was detected by incubating Opal Polymer HRP, Mouse plus Rabbit (AKOYA) for 30 minutes, followed by Opal 520 Reagent (AKOYA). DAPI (AKOYA) was used to counterstain the nuclei. pAKT and pan-CK staining was done as described above, with pAKT (Ser473) (1:200, Cell Signaling Technology, 4060) and pan-CK (1:200, Abcam, ab7753) stained with Opal 570 and Opal 520 Reagent (AKOYA), respectively. The sections were imaged using Vectra Polaris imaging system (Perkin Elmer). Analysis to segment and quantify the cells based on their immunostaining were performed using inForm versions 2.2 (Akoya Biosciences).
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