Multispectral imaging (MSI) was performed using the basic protocol described in Wickenhauser et al. [66 (link)]. Two multiplex panels with different mAbs and opal-dye combinations were used. The first panel included anti-PD-L1 clone E1L3N (Cell Signaling E1L3N, 1:150) in combination with Opal690, anti-Foxp3 clone 236A/E/ (Abcam, 1:00) with Opal540, anti-CD3 clone SP7 (ThermoFisher SP7, 1:100) with Opal570, anti-CD163 clone MRQ-26 (Cell Marque, 1:50) with Opal620 and anti-panCK Ab AE1/AE3 (Dako, 1:150) with Opal520. The second panel comprised anti-TIM-3 (Abcam, ab241332, 1:1000) with Opal 520, anti-PD-1 (Biocare Medical, NAT105, 1:50) with Opal 540, anti-CD8 (DAKO, C8/144b, 1:50) with Opal 570, anti-TIGIT (Biozol, USC-PAN056HU01-1, 1:50) with Opal 620, anti-CD69 (Abcam, ab233396, 1:50) with Opal 650 and anti-HLA-G (Abcam, clone 4H84, 1:100) with Opal 690. After counterstaining with DAPI (Akoya Biosciences, Marlborough, MA), the sections were mounted and scanned with the Vectra Polaris System (Akoya Biosciences, Marlborough, MA) and a mean of 18 regions of interest (ROIs) per slide were taken with a 20 × zoom. The inForm software (Version 2.4.10, Akoya Biosciences) was employed to perform cell segmentation and phenotyping. PhenoptrReports scripts were used within R to evaluate the frequency and density of the different cell types as well as their interspatial relationships.
Vectra polaris system
Manufactured by Akoya Biosciences
Sourced in United States
The Vectra Polaris System is a multispectral imaging platform designed for high-plex tissue analysis. The system captures images of up to 8 fluorescent markers simultaneously, enabling the visualization and quantification of multiple cellular and molecular targets within a single tissue section.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (3 mentions)
Inform automated image analysis software, by Akoya Biosciences (2 mentions)
Anti cd3, by Biocare Medical (2 mentions)
Ab233396, by Abcam (1 mentions)
Anti cd3 clone sp7, by Thermo Fisher Scientific (1 mentions)
Anti pd l1 clone e1l3n, by Cell Signaling Technology (1 mentions)
E1l3n, by Cell Signaling Technology (1 mentions)
Inform software, by Akoya Biosciences (1 mentions)
Tissue microarray, by Shanghai Outdo Biotech Co. (1 mentions)
Anti cd4, by Biocare Medical (1 mentions)
Anti cd31, by Abcam (1 mentions)
6 protocols using vectra polaris system
1
Multiplex Immunofluorescence Imaging of Tumor Microenvironment
2
Histological Evaluation of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About 300 mg of tissue samples were taken as wedge biopsy prior to perfusion (0th hour) and at the end of perfusion (6th hour). Tissue samples were fixed in 10% neutral buffered formalin (Scigen, Gardena, CA) for 24-36 hours prior to embedding in paraffin. Sections were stained with hematoxylin and eosin for qualitative evaluation for sinusoidal and portal triad structural integrity.
Sections were also stained with anti-CD3 (BioCare medical, Pacheco, CA), CD4 (BioCare Medical), DAPI (Akoya Biosciences, Marlborough, MA) and FOXP3 (BioCare medical) (Supplemental Table 2
Sections were also stained with anti-CD3 (BioCare medical, Pacheco, CA), CD4 (BioCare Medical), DAPI (Akoya Biosciences, Marlborough, MA) and FOXP3 (BioCare medical) (
3
Multiplex Immunohistochemistry Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed the staining manually using the same primary antibodies as were used for IHC analysis and those detecting certain other proteins: Clostridia, CD8, C-X3-C motif chemokine receptor 1 (CX3CR1), killer cell lectin like receptor G1 (KLRG1), PD-1, and CD101 (for TCF-1−CX3CR1highKLRG1+PD1−CD101− T effector cells (Teff) and TCF-1−CX3CR1lowKLRG1−PD1+CD101+ terminal T exhausted cells (Tex)), and CD11b, CD14, CD15, CD33, and human leucocyte antigen DR (HLA-DR) (for CD11b+CD14+CD15–CD33+HLA-DR−/lo, monocytic myeloid-derived suppressor cells, M-MDSCs). Binding of the antibodies was detected using the Opal Polymer HRP Ms + Rb immunohistochemistry detection reagent (Perkin Elmer, USA).
The same protocol of IHC was used for the continuous staining, and the next antibody was applied only after complete detection of the previous marker. Primary antibody detection used the Opal Polymer HRP Ms + Rb detection reagent and Opal 7-Color Manual IHC, including six reactive fluorophores: Opal 480, 520, 570, 620, 690, and 780. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) according to the manufacturer's manual. The same protocols were used to stain the positive and negative control groups. The multiplex-stained sections were scanned using the Vectra Polaris system (Akoya Biosciences, USA).
The same protocol of IHC was used for the continuous staining, and the next antibody was applied only after complete detection of the previous marker. Primary antibody detection used the Opal Polymer HRP Ms + Rb detection reagent and Opal 7-Color Manual IHC, including six reactive fluorophores: Opal 480, 520, 570, 620, 690, and 780. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) according to the manufacturer's manual. The same protocols were used to stain the positive and negative control groups. The multiplex-stained sections were scanned using the Vectra Polaris system (Akoya Biosciences, USA).
4
Multiplex Immunofluorescence Profiling of Tumor Microenvironment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue microarrays (Shanghai Outdo Biotech CO.LTD) were deparaffinized using xylene and a gradient ethanol solution. Antigen repair was performed by heating with antigen repair solution. Endogenous peroxidase activity was neutralized with an endogenous peroxidase blocking solution, and the binding of irrelevant antibodies was blocked with BSA. Primary antibodies used in this study included VDR, CD163, CD86, CD68, CCL20, and PanCK. Following primary antibody staining, tissues were stained with Opal polymerized HRP anti-mouse/rabbit secondary detection antibodies. Tissues were then incubated with one of the following fluorophores, Opal Polaris 520, Opal Polaris 570, Opal Polaris 620, Opal Polaris 690, or Opal Polaris 780, according to the manufacturer’s instructions (dilution 1:100). Tissue microarrays were mounted in ProLong Gold antifade reagent with DAPI. Whole tissue sections were scanned at 20x magnification using a Vectra Polaris system (Akoya Biosciences, USA) to capture the stained images. To analyze the spatial distribution of CD68+, CD163+, and CD86 + cells in the area surrounding the tumor, an algorithm was designed to create a 200 μm-thick band outside the tumor margin. The peritumoral compartment was defined as the region outside the tumor margin within 200 μm.
5
Multispectral Imaging of Tumor Metastases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Imaging was performed with the Vectra Polaris system (Akoya Biosciences, Marlborough, MA, USA). First, each whole slide was scanned at 10× magnification. Then, the regions (1.86 × 1.39 mm) for image acquisition were selected to capture areas from the central part of the metastases and from the periphery. The selected regions were imaged in a multispectral mode at a resolution of 2 pixels per 1 μm.
6
Quantifying T Regulatory Cells in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were fixed in 10% neutral buffered formalin (Scigen, Gardena, CA) for 24–36 h prior to embedding in paraffin. Sections were then stained with hematoxylin and eosin to evaluate airway and alveoli structural integrity and anti‐CD31 (Abcam, Cambridge, UK) to evaluate vasculature structural integrity.
To quantify T regulatory cells, sections were stained with anti‐CD3 (BioCare medical, Pacheco, CA), anti‐CD4 (BioCare Medical), DAPI (Akoya Biosciences, Marlborough, MA) and FOXP3 (BioCare medical) (Supplemental table3 ), and slides were scanned by Vectra Polaris System (Akoya Biosciences) and analyzed with Inform Automated Image Analysis Software (Akoya Biosciences). A minimum of five region of interests (ROI) (931 um × 698 um) were selected within each slide, in which number of tissue regulatory T cells (Treg cells) and CD3+ T cells were counted. The percentage of Treg cells out of all CD3+ T cells was calculated for each ROI.
To quantify T regulatory cells, sections were stained with anti‐CD3 (BioCare medical, Pacheco, CA), anti‐CD4 (BioCare Medical), DAPI (Akoya Biosciences, Marlborough, MA) and FOXP3 (BioCare medical) (Supplemental table
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!