Sections of 3 µm were deparaffinized, rehydrated, and heat-mediated antigen retrieved with citrate buffer (pH 9). Staining with primary antibodies (rabbit anti-mouse AGE polyclonal antibodies (1:400), Bioss Antibodies, Woburn, MA, United States) and DAPI was done with opal fluorochrome–based staining chemistry AKOYA Opal 4-Color Anti-Rabbit Manual IHC Kit (Phenoptics™/Akoya Biosciences, Marlborough, MA, United States). The images were taken using the multispectral scanner Vectra Polaris™ (Akoya Biosciences) at the Institute of Forensic Medicine, Pathology Department, University Hospital Jena, visualized with the software Phenochart™ 1.1 (Akoya Biosciences, Marlborough, MA, United States), and quantified using a score of 0–5 (0 = no accumulation; 1 = weak accumulation, 2 = mild accumulation, 3 = moderate accumulation, 4 = strong accumulation, and 5 = very strong accumulation).
Phenochart 1
Manufactured by Akoya Biosciences
Sourced in United States
Phenochart™ 1.1 is a digital pathology software tool developed by Akoya Biosciences. It is designed to facilitate the visualization and analysis of whole-slide images obtained from tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Vectra polaris, by Akoya Biosciences (3 mentions)
Vectra polaris imaging system, by Akoya Biosciences (2 mentions)
Roti block, by Carl Roth (1 mentions)
Inform2, by Akoya Biosciences (1 mentions)
Opal 7 color automation ihc kit, by Akoya Biosciences (1 mentions)
Cxcl13, by Thermo Fisher Scientific (1 mentions)
H 3401, by Vector Laboratories (1 mentions)
S1699, by Agilent Technologies (1 mentions)
Ba 2020, by Vector Laboratories (1 mentions)
Vectra polaris slide scanner, by Akoya Biosciences (1 mentions)
Vectra polaris, by PerkinElmer (1 mentions)
Az100 multizoom microscope, by Nikon (1 mentions)
Nis elements imaging software, by Nikon (1 mentions)
Vector blue, by Vector Laboratories (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Bright dab, by Immunologic (1 mentions)
Vectra 3, by PerkinElmer (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
10 protocols using phenochart 1
1
Quantitative Immunofluorescence Assay for AGE
2
CTGF Immunohistochemistry Staining Protocol
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For CTGF staining, 3 µm sections were deparaffinized and rehydrated. No heat mediated antigen retrieval was performed. Blocking of endogenous peroxidase was achieved by incubation with 3% H2O2 (Roth, Karlsruhe, Germany) for 10 min at room temperature. Following the blocking with Roti-Block (Roth, Karlsruhe, Germany), the sections were incubated with primary rabbit polyclonal anti-mouse CTGF antibodies (Abcam, Cambridge, United Kingdom) overnight at 4°C. After incubation with peroxidase-labeled goat anti-rabbit IgG antibody (SeraCare, Milford, MA, United States), diaminobenzidine (DAB) (DAB-peroxidase substrate kit; Vector Laboratories, Burlingame, CA, United States) was used as a chromogen. Whole-slide images were taken using the multispectral scanner Vectra Polaris™ (Akoya Biosciences) by Pathology Department, University Hospital Jena, visualized with software Phenochart 1.1 (Akoya Biosciences, Marlborough, MA, United States) and quantified with a score of 0–5 (0 = no fibrosis; 1 = weak fibrosis, 2 = mild fibrosis, 3 = moderate fibrosis, 4 = strong fibrosis, and 5 = very strong fibrosis).
3
RGB Snapshot Analysis in Phenochart
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RGB (red green blue) snapshots were acquired in Phenochart 1.0.12 (Akoya Biosciences). Image analysis was performed using the open source program Fiji ImageJ version 1.52p.
4
Automated Tumor Immune Profiling
5
Multiparametric Immunofluorescence Profiling of FFPE Tissue
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FFPE tissue sections of 4 μm were cut and prepared for immunofluorescence staining using the following combination of antibodies against CD14 (clone EPR3653, Cell Marque, detection by Opal 570), FDC (clone CNA.42, Invitrogen, detection with Opal 690), CXCL-13 (rabbit polyclonal, ThermoFisher, detection with Opal 520), IL-17 (goat polyclonal, R&D systems, detection with Opal 620), CD20 (clone L26, ThermoFisher, detection with Opal 480) and DAPI (nuclear marker), as previously described.38 (link) Briefly, the staining consisted of consecutive rounds of antigen retrieval, staining with primary antibody, secondary HRP-labeled antibody and detection with optimized fluorescent Opal tyramide signal amplification (TSA) dye (Opal 7-color Automation IHC kit, from Akoya, Ref. NEL821001KT) and repeated antibody denaturation cycles. Multispectral images were acquired using the latest Vectra Polaris imaging system from Akoya. All images were recorded using a 20× magnification. The Phenochart 1.0.12 software (Akoya), a whole-slide contextual viewer was used for identification of regions of interest and acquisition of unmixed multispectral images.
6
Multispectral Imaging of FFPE Tissues
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Multispectral images (MSI) were acquired using the latest Vectra Polaris imaging system from Akoya. All images were recorded using a 20x magnification. The Phenochart 1.0.12 software (Akoya), a whole-slide contextual viewer with annotation capability was used for navigation around slides and identification of Regions Of Interest (ROIs), for high-resolution multispectral acquisition. A spectral library containing the emitting spectral profile of all six reporter fluorophores (in monoplex staining) and of DAPI, plus the autofluorescence signal present in FFPE tissues, obtained from a target tissue of relevant slide, was created with the Nuance Image Analysis software (Akoya). The subsequent imaging and analysis were performed on selected annotations and specific MSI fields, representing the 30–40% of the tissue. The whole tissue slides were pre-scanned at a 10x magnification and approximately 20–40 regions were selected for the acquisition of high-power (20x) MSI.
7
Immunohistochemical Staining of α-Dystroglycan
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Immunohistochemical (IHC) staining was carried out on 2 μm formalin-fixed, paraffin-embedded sections using standard procedures. In brief, antigen retrieval was achieved with target retrieval solution (S1699, Agilent Technologies) via microwave heating. Incubation with the primary antibody IIh6 (Santa Cruz) at a concentration of 4 μg/ml was done at room temperature for one hour. As a detection system, biotinylated anti-mouse IgM (BA2020, Vector Labs) and streptavidin-HRP (RE 7104, Novocastra, Newcastle, UK) was used. Samples were developed via exposure to 3,3`-diaminobenzidine (DAB+, K3468, Agilent) and counterstained with hematoxylin Gill’s Formula (H-3401, Vector Labs). For IHC staining against α-DG on mouse FFPE tissue, Crystal MausBlock (Fa. DCS, Hamburg, Germany, ML125R015) was used to avoid non-specific binding of the secondary antibody. Processed slides were scanned on a Vectra Polaris™ slide scanner using 40-fold scan resolution and snapshots taken via Phenochart 1.0.8 software (both AKOYA Biosciences, MA, USA).
8
High-Resolution Quantitative Imaging Workflow
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Processed slides from immunohistochemical analysis were scanned using the quantitative slide scanner Vectra Polaris (Perkin Elmer, Waltham, MA). Scanning was performed using the highest possible instrument setting (40-fold scan resolution). Snapshots of these scans were taken via Phenochart 1.0.8 software (Akoya Biosciences, Marlborough, MA). PDTO images were generated using an AZ100 multizoom microscope (Nikon, Minato City, Japan) and NIS Elements Imaging Software (version 5.00.00; Nikon).
9
Multiplex Immunohistochemical Staining
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Nonspecific binding was blocked with Blocking One (Nacalai Tesque) for 30 min at room temperature. A sequential double staining procedure50 (link) was initiated with CitFbg, and was visualized by BrightVision HRP-conjugated anti-mouse IgG polymer (DPVM-HRP, Immunologic) and Bright DAB (BS04, ImmunoLogic). The DAB reaction product effectively sheltered the first set of antibodies. Then, the process was continued with VE-cadherin (1:200, sc6458, Santa Cruz) and anti-human SAA1/2 (1:1000, ab207445, Abcam) and anti-human Fbg (1:1000, A0800, Dako), and was visualized by BrightVision alkaline phosphatase (AP-)conjugated anti-rabbit IgG polymer (DPVR-AP, ImmunoLogic) and Vector Blue (SK-5300, Vector Laboratories; Burlingame, CA). The slides were counterstained, washed with tap water, and coverslipped with PathodMount (Fujifilm Wako) after dehydration with Clear-Rite 3TM (Richard-Allen Scientific LLC, USA). Images were acquired with Vectra 3 (PerkinElmer), Phenochart 1.1.0, and inForm 2.4.9 (Akoya Bioscience).
10
Quantification of Renal Fibrosis via Masson's Trichrome
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As described above, the paraffin-embedded tissues were sliced at 3 µm thickness, dewaxed, and rehydrated. Masson’s trichrome staining was then performed using the standard protocol. Whole-slide images were taken of the sections using the multispectral scanner Vectra Polaris™ (Akoya Biosciences, Malborough, MA, USA) and visualized by the software Phenochart 1.1.0 (Akoya Biosciences, Marlborough, MA, USA). For the quantification of renal glomerular and tubular fibrosis, Masson’s staining was visually assessed for collagen blue staining in the glomeruli and in the tubulointerstitium, and quantified with a score of 0–5 (0 = no blue staining; 1 = <10%, 2 = 10–20%, 3 = 20–40%, 4 = 40–60%, and 5 = >60%).
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