Human PDAC samples (n = 6) were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer sections were used for the immunofluorescence staining. Immunofluorescence staining on FFPE tissue was performed using the Opal 7-Color Automated Immunohistochemistry Kit (cat: NEL821001KT, Akoya Biosciences). A multiplex panel of immune markers was developed with antibodies against: CXCR4 (clone EPUMBR3, cat: ab181020, dilution 1:300, Abcam), CD8 (clone C8/144B, cat: M710301–2, dilution 1:200, Dako/Agilent), CD68 (clone PG-M1, cat: M087601–2, dilution 1:400, Dako/Agilent), Cytokeratin (Cytokeratin 7, clone OV-TL, cat: M701801–2, dilution 1:500, Dako/Agilent + Cytokeratin 19, clone A53-B/A2.26, cat: 760–4281, RUI, Cell Marque/Roche). The staining procedure was performed using an automated staining system (BOND-RX; Leica Biosystems). All markers were sequentially applied and paired with respective Opal fluorophores. To visualize cell nuclei, the tissue was stained with 4‘,6-diamidino-2-phenylindole (spectral DAPI, Akoya Biosciences). Stained slides were scanned 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. Spectral unmixing, multispectral image acquisition was carried out using the InForm Tissue Analysis Software version 2.4.10 (Akoya Biosciences).
Mantra 2 quantitative pathology workstation
Manufactured by Akoya Biosciences
Sourced in United States
The Mantra 2 Quantitative Pathology Workstation is a multiplex imaging system designed for the analysis of formalin-fixed, paraffin-embedded tissue samples. The system enables the simultaneous detection and quantification of multiple protein targets within a single tissue section.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 clone c8 144b, by Agilent Technologies (2 mentions)
Cd68 clone pg m1, by Agilent Technologies (2 mentions)
Bond rx, by Leica (2 mentions)
Inform tissue analysis software, by Akoya Biosciences (1 mentions)
Opal 6 plex detection kit, by Akoya Biosciences (1 mentions)
Cd4 clone ep204, by Cell Marque (1 mentions)
Cytokeratin clone ae1 ae3, by Thermo Fisher Scientific (1 mentions)
4 protocols using mantra 2 quantitative pathology workstation
1
Multiplex Immunofluorescence Profiling of PDAC
2
Quantitative Pathology Imaging Workflow
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Tissues were imaged with a Mantra 2 Quantitative Pathology Workstation (Akoya Biosciences, Marlborough, MA, USA) with a 40x objective. Six representative images were taken per tissue. Area not containing tissue and prostate lumens were removed to obtain total tissue area to normalize positive cell counts that were manually counted. Both experimental groups were represented on each tissue blocks/slides, and slides from the same tissue and timepoint were processed together.
3
Quantitative Pathology Imaging of Tissue Samples
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IHC tissues were imaged with a Mantra 2 Quantitative Pathology Workstation (Akoya Biosciences, Marlborough, MA, USA) with a 20x objective. Three representative images were taken per tissue. Area not containing tissue was removed (CD45+ and OPN), or only the stroma was selected (for COL1A1 density) with inForm software using trainable tissue segmentation. Optical density (OD) or number of positive cells was calculated by the software. CD45-positive cells were counted by hand in the chronic inflammation group. Cell counts were normalized to tissue area.
RNAscope staining was captured with a 40× objective (6 images/tissue), and the staining area was determined using the Inform software thresholding function. Individual particle size (averaged 0.8 micron) was used to calculate particle number from the total positive area and normalized to tissue area.
All four experimental groups were represented on each tissue blocks/slides, and slides from the same tissue and timepoint were processed together. The imaging was not blinded to allow the correct identification of tissues on arrays.
RNAscope staining was captured with a 40× objective (6 images/tissue), and the staining area was determined using the Inform software thresholding function. Individual particle size (averaged 0.8 micron) was used to calculate particle number from the total positive area and normalized to tissue area.
All four experimental groups were represented on each tissue blocks/slides, and slides from the same tissue and timepoint were processed together. The imaging was not blinded to allow the correct identification of tissues on arrays.
4
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).
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