Formalin-fixed paraffin-embedded sections were placed slides, deparaffinized, and incubated with STAG2 antibody (Cell Signaling Technology, catalog no. 5882, RRID:AB_10834529), Ki67 antibody (Abcam, catalog no. ab15580), or Cleaved Caspase 3 (Cell Signaling Technology, catalog no. 9664). Biotinylated anti rabbit (Vector BA-1000) was applied for 30 minutes followed by Elite ABC (Vector PK6100) for 30 minutes. DAB (diaminobenzidine; Dako; catalog no. K3468) was applied for 5 minutes for visualization. Slides were counterstained with hematoxylin then dehydrated, cleared, and cover slipped. TMA and IHC slides were digitally scanned using Aperio Scanscope (Aperio Technologies, Inc.) with 20× bright-field microscopy. These images were then accessible using Spectrum (Aperio Technologies, Inc.), a web-based digital pathology information management system. Aperio ImageScope version 11.2.0.780 (Aperio Technologies, Inc., RRID:SCR_014311) was used to view and analyze images. For additional details, see Supplementary Materials and Methods .
Aperio imagescope version 11
Manufactured by Leica
Sourced in United States
Aperio ImageScope version 11.2.0.780 is a digital pathology viewer software application developed by Leica Biosystems. It is designed to display and analyze digital images of tissue samples captured using Leica's digital slide scanners.
Automatically generated - may contain errors
Lab products found in correlation
Aperio scanscope, by Leica (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Ki67 antibody, by Abcam (2 mentions)
Abc elite, by Vector Laboratories (2 mentions)
Stag2 antibody, by Cell Signaling Technology (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Envision horseradish peroxidase system, by Agilent Technologies (2 mentions)
Dako autostainer, by Agilent Technologies (2 mentions)
Aperio scanscope xt, by Leica (2 mentions)
Dpx mountant, by Merck Group (2 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Ma5 14520, by Cell Signaling Technology (1 mentions)
Hpa001520, by Merck Group (1 mentions)
Aperio at2, by Leica (1 mentions)
Integrator system, by Visiopharm (1 mentions)
Bond polymer refine red detection kit, by Leica (1 mentions)
Aperio cs2, by Leica (1 mentions)
8 protocols using aperio imagescope version 11
1
Immunohistochemistry of STAG2, Ki67, and Cleaved Caspase 3
2
Automated Digital Pathology Analysis of Immune Markers
3
Automated IHC and Digital Pathology Analysis
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IHC assays and automated digital pathology analysis were performed on formalin-fixed paraffin-embedded tissue blocks. Tissue sections (4–5 μm) were prepared on slides loaded on a DAKO autostainer (Dako) and after serum-free protein block the respective primary antibodies for CD3, CD4, CD8, Foxp3, IDO1, PD-1, and PD-L1 were applied separately. The EnVision+ horseradish peroxidase system (Dako) and DAB (3,3-diaminobenzidine) chromogen were used for visualization. Slides were digitally scanned using Aperio Scanscope (Aperio Technologies, Inc.) with 20 × bright-field microscopy. These images were then accessible using Spectrum (Aperio Technologies, Inc.), a web-based digital pathology information management system. Slide images are automatically associated to a digital slide created in the Digital Slide table in Aperio eSlide Manager. Once slides are scanned, Aperio ImageScope version 11.2.0.780 (Aperio Technologies, Inc.) was used to view images for image analysis. An outline of the tumor and the size of the analysis area were defined, and the lymphocytes were counted using an optimized algorithm for each stain and results were normalized to number of lymphocytes per square millimeter.
4
Immunohistochemical Analysis of STAG2, Ki67, and Cleaved Caspase 3
5
Automated Tissue Analysis of T-cells
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Slides were scanned using a high-resolution digital scanner (Aperio Scanscope XT; Leica Biosystems, North Ryde, NSW, Australia) at × 40 magnification. Evaluation of T-cell density (cells per mm2 tissue) was performed using image analysis software (Aperio Imagescope version 11; Leica Biosystems) after careful manual annotation of the cores to ensure that the area of analysis matched the histology type to which the core was assigned, and to exclude areas of damaged or missing tissue. Data presented represent the average of two cores where possible.
6
Quantifying T-cell Density in Gastric Tumor
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Images of the gastric tumor sections (4-µm) were captured using a high-resolution digital scanner (Aperio Scanscope XT; Leica Biosystems, North Ryde, NSW, Australia) at ×40 magnification. The T-cell density (cells per mm2 tissue) was analyzed using software (Aperio Imagescope version 11; Leica Biosystems).
7
Immunohistochemistry for Tumor Markers
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IHC for Ki-67 (Invitrogen—MA5-14520, 1:200), cleaved caspase 3 (Cell Signaling), and ATP5B (Sigma-Aldrich—HPA001520, 1:500) was performed on 4-μm FFPE sections produced from the xenograft tumours using commercial equipment and a DAB detection kit. Slides were counterstained with hematoxylin and mounted using DPX mountant (Sigma-Aldrich, St. Louis, MO, USA). Slides were scanned with an Aperio AT2 digital slide scanner (Leica Biosystem, Milton Keynes, United Kingdom) with a 20x lens and analyzed with imaging software (Aperio Image Scope version 11.2; Leica Biosystems, Inc., Buffalo Grove, IL, USA). Automated digital image analysis was performed using the Visiopharm Integrator System (Visiopharm, Hoersholm, Denmark). H-Score and percentage of positive cells were used as the image analysis output for cleaved caspase-3 and ATP5B expression. Percentage of positive cells was used as the image analysis output for Ki-67 expression. H-Score was calculated using the following formula: [1 × (% of weakly positive cells) + 2 × (% of moderately strong positive cells) + 3 × (% strong positive cells)].
8
Immunohistochemistry Protocol for CysLT1 and CysLT2
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IHC for CysLT1 (Abcam, Cambridge, UK [ab151484], 1:200) and CysLT2 (Cayman Chemical, Ann Arbor, MI, USA [CAY120560], 1:500) was performed on 4-μm FFPE sections arranged on the above mentioned TMA using commercial equipment (Leica Bond RXm System; Leica Microsystems Ltd., Milton Keynes, UK) and a detection kit (Bond Polymer Refine Red Detection Kit; Leica Biosystems, Inc., Buffalo Grove, IL, USA) as previously described [73 (link)]. Slides were counterstained with hematoxylin and mounted using DPX mountant (Sigma-Aldrich, St. Louis, MO, USA). Colorectal cancer tissue served as the positive control (Figure S2A ); negative control was omission of the primary antibody (Figure S2A ). Slides were scanned using a slide scanner (Aperio CS2; Leica Biosystems, Inc., Buffalo Grove, IL, USA) and analysed with imaging software (Aperio Image Scope version 11.2; Leica Biosystems, Inc., Buffalo Grove, IL, USA). Each core was scored based on intensity (0, 1, 2, or 3) and percentage of tumour cells stained. The final score was calculated using the following equation: (scoring intensity × % of cells stained)/n number of samples [73 (link)]. The IHC-stained slides were scored by three independent investigators (S.E.C., H.K., K.S.). Melanoma-specific survival is defined as death from metastatic melanoma. Overall survival is defined as death by any cause.
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