Multicolor immunofluorescence analyses were performed using 3-μm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissues. Briefly, slides were deparaffinized in xylene and hydrated in a series of decreasing graded ethanol series. After heat-induced antigen retrieval in citrate buffer (pH = 6), samples were permeabilized with 0.5% Triton X-100, blocked with 5% goat serum-phosphate-buffered saline (PBS), and sequentially co-stained with antibodies recognizing VCAM-1 (Abcam, ab134047), CD248 (Abcam, ab217535), 4-HNE (Abcam, ab46545), FAPα (Invitrogen, BMS168; Abcam, ab218164), Mfap4 (Thermo, PA5-24865), Sparcl1 (Santa Cruz, sc-514275), F4/80 (Cell Signaling Technology, #70076), rabbit IgG Isotype Control (Invitrogen, 31235), mouse IgG Isotype Control (Invitrogen, 14-4714-82), and DAPI. A TSA indirect kit (PerkinElmer) was used according to the manufacturer’s instructions. Vectra® Polaris™ Imaging System (Akoya Biosciences) was used to collect images, and image analysis was performed using HALO™ Image Analysis Software.
Vectra polaris imaging system
Manufactured by Akoya Biosciences
Sourced in United States
The Vectra Polaris imaging system is a multispectral imaging platform designed for the analysis of tissue samples. It provides high-resolution, multiplexed images of multiple biomarkers within a single tissue section. The system utilizes advanced imaging and computational technologies to capture and analyze complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Halo software, by Indica Labs (3 mentions)
Inform software, by Akoya Biosciences (2 mentions)
Phenochart, by Akoya Biosciences (2 mentions)
Phenochart 1, by Akoya Biosciences (2 mentions)
Halo image analysis platform, by Indica Labs (2 mentions)
Discovery xt processor, by Roche (2 mentions)
F4 80, by Cell Signaling Technology (1 mentions)
Tsa indirect kit, by PerkinElmer (1 mentions)
Ab134047, by Abcam (1 mentions)
Rabbit igg isotype control, by Thermo Fisher Scientific (1 mentions)
Mouse igg isotype control, by Thermo Fisher Scientific (1 mentions)
Ab46545, by Thermo Fisher Scientific (1 mentions)
Opal 7 color ihc kit, by Akoya Biosciences (1 mentions)
Ab86734, by Abcam (1 mentions)
Ab9498, by Abcam (1 mentions)
P0448, by Agilent Technologies (1 mentions)
Ab217344, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Inform software v2, by Akoya Biosciences (1 mentions)
Inform image analysis software, by Akoya Biosciences (1 mentions)
27 protocols using vectra polaris imaging system
1
Multicolor Immunofluorescence of FFPE Tissues
2
Multiplex IF Staining of TMAs
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Tissues of tissue microarray (TMA) were sliced into sections with 9 µm thickness and mounted onto glass slices. Multiplex immunofluorescent staining was performed on TMAs using Opal 7‐Color IHC kits (Akoya Biosciences, Hopkinton, MA) according to the manufacturer's instructions. Briefly, sections were deparaffinized using xylene and progressively hydrated by ethanol ending with a distilled water wash. Microwave treatment was performed in AR6 buffer for antigen retrieval. Then slides were sequentially stained with the following primary antibodies and fluorescent dyes: anti‐CD31 (1:50, #ab9498, Abcam)/Opal‐520, Anti‐phospho‐FGFR4 (1:200, #PA5105531, Invitrogen)/Opal‐620, anti‐pan‐cytokeratin (1:100, #ab86734, Abcam)/Opal‐690. For each molecule being detected, slides were incubated with the specific primary and secondary antibody, followed by Opal fluorophore solution and incubated for 10 min at room temperature. Afterward, microwave treatment was performed again to remove the specific 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 coverslips. Digital images of all cores of TMA were acquired using the Vectra Polaris Imaging System and analyzed by inForm software (Akoya Biosciences).
3
CD8+ T Cell Quantification in Murine Tumors
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C57BL/6 mice bearing MC38 tumors (average volume of 80 mm3) were treated with muC5H9v2 mAb, CX-630 Pb-Tx, or isotype control (5 mg/kg) on days 0, 3, and 7. On day 8, tumors were excised, fixed in formalin, and embedded in paraffin blocks. CD8 immunohistochemical analysis was performed by Cureline, Inc. Briefly, 5 μm sections were cut, deparaffinized, and rehydrated followed by antigen retrieval using an EDTA buffer. Sections were washed and incubated in a blocking solution. Primary staining was performed with a 200-fold dilution of rabbit anti-CD8 antibody (Abcam, catalog #ab217344) or isotype control (Abcam, catalog #ab172730) in staining buffer (PBS with 0.5% Tx-100 and 1% FBS) for 1 hour at room temperature. After several washes, tissue sections were stained with an anti-rabbit HRP-conjugated secondary antibody for 20 minutes at room temperature (Dako, catalog #P0448). After a final wash step, the slides were incubated with a 3,3′-diaminobenzidine chromogen for 10 minutes at room temperature. After rinsing, slides were counterstained with hematoxylin, dehydrated, cleared, and mounted. Brightfield whole-tissue scans where performed on the slides using the Vectra Polaris Imaging System (Akoya Biosciences) at a 20× magnification. Snapshots images were captured digitally from the scan at 10× magnification using Phenochart software version 1.0.
4
Multiplex Fluorescent Staining and Tissue Analysis
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Tumor tissue sections that underwent multiplex fluorescent staining for each fluorophore were imaged using the Vectra Polaris imaging system (Akoya Biosciences) under the appropriate fluorescent filters (green for Opal 520, red for Opal 620 and DAPI) in order 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 (Akoya Biosciences) and imaging at 20× power for further analysis. Analysis of the multispectral images was conducted using inForm image analysis (Akoya Biosciences). Representative images of each sample used to establish tissue segmentation and cell segmentation algorithms were applied to batch analysis of all high power multispectral images. The software was then trained to segment the tissue categories into the cell components: the nuclei, cytoplasm and membrane of each cell. The positivity threshold of each marker was then determined and recorded for further data analysis. Once the algorithm was completed, all images were imported into inForm, and run as a batch.
5
Automated Phenotyping of Tissue Sections
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Imaging was performed with the VectraPolaris imaging system (Akoya Bioscience), and images were analyzed by using the phenotyping application of the inForm software V2.4.10 (Akoya Bioscience). Phenotype classification was performed automatically by the written algorithm using inform Software. Multispectral image analysis was performed with Phenochart, InForm Image analysis software (Akoya Biosciences Inc.), and HALO software (Indica labs). For distance analysis in HALO software 5 slides of the infarcted regions, 5 slides of the non-infarcted regions and 5 healthy controls were examined, calculating proximity distances from one population to the other and vice-versa in a delimited radius range.
6
Multiplex Immunohistochemistry for Biomarker Analysis
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mIHC/IF was performed using an Opal 7-color Manual IHC Kit (PerkinElmer, USA), as previously described in other studies [18 (link), 19 (link)]. Tissue sections (4 µm thick) were labeled with primary antibodies against EGFR, Nestin, Sox2, and CADM1, followed by appropriate secondary antibodies. All antibodies used were listed in Supplementary Table 3 . The slides were mounted with ProLong Gold Antifade Reagent containing DAPI, and scanned using Vectra® Polaris™ Imaging System (Akoya Biosciences). Images were analyzed by Image J software (National Institutes of Health, USA).
7
Histomorphometric Analysis of Bone Samples
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Bone histomorphometry was performed on both paraffin sections. In brief, the femur specimens, after fixation in 4% PFA for 24 h, were decalcified with 10% ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich) for 4 weeks. For paraffin sections, the specimens were processed, embedded in paraffin, and cut into 5-µm-thick sections using a rotary microtome (RM215, Leica). H&E staining, TH staining (AB152, Millipore), and TRAP staining (MK30, Takara) were performed on selected sections from each sample following the manufacturer’s instructions. Images were captured using the Vectra Polaris Imaging System (Akoya Biosciences) and analyzed using ImageJ (NIH).
8
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.
9
Multispectral Fluorescent Imaging Analysis
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Slides were scanned with the Vectra Polaris imaging system (Akoya Biosciences) following the manual’s instructions, with high-power field scan (×40) using the fluorescent mode. The microscope captured the multispectral fluorescent spectra separately at the corresponding tyramide Opal fluorophore wavelength, with preset exposure times, and then these captures were stacked in 1 image (QPTiff) without disrupting the unique fluorescent spectral signature of the markers. The QPTiff image was analyzed in Visiopharm software for the 3 regions of interest: necrosis, tumor, and brain-tumor interface/infiltrating edge.
10
Immunohistochemical Analysis of Brain and Intestine
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For immunohistochemistry, tissues (brains and intestines) were sectioned at 30 μm thickness and treated with methanolic H2O2 for 30 min. The sections were incubated with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min and blocked with 4% normal serum in PBS for 15 min before incubating with the primary antibody. The sections were incubated overnight with TH (1:2,000, Servicebio, GB11181), ZO-1 (1:300, Servicebio, GB111981), occludin (1:500, Servicebio, GB111401), or claudin (1:300, Servicebio, GB11032). The sections were washed three times in PBS (pH 7.4, and incubated with secondary antibodies (1:200, Servicebio, GB23303) for 50 min at room temperature. After the slices were slightly spin-dried 3 times, 3,3′-diaminobenzidine tetrahydrochloride (DAB) condensed chromogen (Servicebio, G1211) was utilized to visualize. After immunostaining, sections were counterstained with hematoxylin (Servicebio, G1004). Sections were photographed using the Vectra Polaris Imaging System (Akoya).
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