TMAs were prepared from patients with metastatic CCRCC enrolled in phase II trials at the U.T.M.D. Anderson Cancer Center and were treated with sorafenib or sorafenib plus interferon (25 (link)). Three cores were obtained per patient and stained using HAF monoclonal antibody (20 (link)). Images were scanned using an Aperio AT2 slide scanner, and HAF nuclear staining was quantitated using Aperio Image analysis software (Leica Biosystems Inc, Buffalo Grove, IL). TCGA data was accessed using OncoLnc (26 ). Both datasets were stratified into two equal groups of high or low HAF transcript or protein. Kaplan Meier survival curves, p-values and hazard ratios were calculated using Prism 7.0.
Aperio image analysis software
Manufactured by Leica
Aperio Image analysis software by Leica is a digital pathology solution designed for the visualization and analysis of high-resolution whole slide images. It enables the efficient viewing, management, and interpretation of digital tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
At2 slide scanner, by Leica (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Bond rx automated system, by Leica (1 mentions)
Anti prb, by Cell Signaling Technology (1 mentions)
Hematoxylin, by Bio-Optica (1 mentions)
Eosin g, by Bio-Optica (1 mentions)
Aperio at2 digital scanner, by Leica (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Imagescope, by Leica (1 mentions)
Halo image analysis, by Indica Labs (1 mentions)
Discovery xt, by Roche (1 mentions)
Cell conditioning solution, by Roche (1 mentions)
Ki 67 primary antibody, by BD (1 mentions)
Aperio cs2 digital pathology scanner, by Leica (1 mentions)
Inhibitor cm, by Roche (1 mentions)
Chromomap dab kit, by Roche (1 mentions)
7 protocols using aperio image analysis software
1
Prognostic impact of HAF in metastatic CCRCC
2
Biomarker Stability in Glioblastoma Progression
3
Histological Analysis of YAP and Ki67 in Kidney Sections
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For histological analysis mice were sacrificed at the indicated time, kidneys were collected, weighted and fixed in formalin 10% (BioOptica, #05-01005Q), included in paraffin and cut 5 μm/slides. Kidney sections were air-dried and rehydrated in PBS (Sigma-Aldrich, #P4417). For YAP staining kidney sections were incubated with YAP antibody and 5 min in Eosin G (BioOptica, #05-10002/L); For Ki67 staining kidney sections were incubated with Ki67 antibody and 2 min in Hematoxylin (BioOptica, #05-06015/L). Sections were then washed, processed through a dehydration alcohol scale and mounted in DPX (Sigma-Aldrich, #06522). For the semi-automated quantification, slides were acquired with Aperio AT2 digital scanner at magnification of 40 × (Leica Biosystems) and analyzed with Imagescope (Leica Biosystem). YAP and Ki67 nuclear staining per cyst was performed by Aperio Image analysis software (Leica Biosystems) and counted manually.
4
Quantitative IHC and IF Analysis
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All organs were fixed in 10% neutral buffered formalin for 48 hours prior to paraffin-embedding and sectioning. All IHC staining was completed on the Leica Biosystems BOND MAX platform (Buffalo Grove, IL), antibodies used for chromogenic IHC staining and tissue immunofluorescence are listed in Supplementary Table S1.
IHC images were acquired using the Aperio AT2 Whole Slide Digital Scanner (Leica Biosystems) at 20× magnification. Quantitative analysis was performed by Aperio Image Analysis Software (Leica Biosystems) employing membrane (E-cadherin, CK19), nuclear (Ki67), and positive pixel count algorithms (collagen, smooth muscle actin).
For phospho-SMAD2/SMA immunofluorescence costaining, 5-μm–thick sections were deparaffinized, followed by 10 minutes of heat-induced epitope retrieval in citrate (phospho-SMAD2/SMA) or EDTA buffer (phospho-STAT3/SMA double staining). Primary antibodies were incubated for 60 minutes and secondary antibodies for 30 minutes with UltraCruz background blocking performed one hour prior. Tissues were manually counterstained with DAPI combined with mounting medium. Fluorescent images were acquired using the Aperio FL Multi-channel Fluorescence Slide Scanner (Leica Biosystems) at 20× magnification, and quantitative analysis was performed by HALO Image Analysis (Indica Labs) utilizing the High-Plex FL v3.0.3 Classifier Algorithm.
IHC images were acquired using the Aperio AT2 Whole Slide Digital Scanner (Leica Biosystems) at 20× magnification. Quantitative analysis was performed by Aperio Image Analysis Software (Leica Biosystems) employing membrane (E-cadherin, CK19), nuclear (Ki67), and positive pixel count algorithms (collagen, smooth muscle actin).
For phospho-SMAD2/SMA immunofluorescence costaining, 5-μm–thick sections were deparaffinized, followed by 10 minutes of heat-induced epitope retrieval in citrate (phospho-SMAD2/SMA) or EDTA buffer (phospho-STAT3/SMA double staining). Primary antibodies were incubated for 60 minutes and secondary antibodies for 30 minutes with UltraCruz background blocking performed one hour prior. Tissues were manually counterstained with DAPI combined with mounting medium. Fluorescent images were acquired using the Aperio FL Multi-channel Fluorescence Slide Scanner (Leica Biosystems) at 20× magnification, and quantitative analysis was performed by HALO Image Analysis (Indica Labs) utilizing the High-Plex FL v3.0.3 Classifier Algorithm.
5
Quantifying Cellular Proliferation in Formalin-Fixed Tissue
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Four μm-thick sections of formalin-fixed paraffin-embedded (FFPE) explant tissue were stained for the proliferative marker Ki67. Automated immunohistochemistry (IHC) was carried out on a Discovery XT staining module (Roche). Antigen was retrieved by incubating slides in Cell Conditioning Solution (Roche). Endogenous peroxidase activity was reduced by incubating slides in Inhibitor CM (Roche). Slides were incubated in Ki67 primary antibody (Novocastra; 1:100) or Anit-AR primary antibody (clone G122-434, BD) for 1 h at 37°C followed by HRP-conjugated Discovery OmniMaP HRP secondary antibody (Roche) for 30 min. A ChromoMap DAB kit (Roche) was used for detection. Slides were counterstained in hematoxylin and mounted in DPX. Stained slides were imaged using an Aperio CS2 Digital Pathology scanner (Leica). Regions of interest (i.e. epithelial cells) were manually annotated and the percentage of Ki67 positive cells was determined using Aperio Image Analysis software (Leica) in a minimum of four representative 40× magnification regions containing a total of at least 1000 nuclei. Positive control tissue for Ki67 is shown in Supplementary Figure S8 alongside no primary control staining. Serial sections were also stained for p63 (basal cell marker identifying benign regions) and AMACR (cancer cell marker) to define regions of cancer (Supplementary Figure S9 ).
6
Quantitative Analysis of Tumor Immunohistochemistry
7
Nuclei Quantification and IHC Analysis
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Individual nuclei counts were counted from H&E sections (normalized to a length of 300 µm) using the Nuclei Seg plugin of the Halo image analysis platform. IHC staining was quantified using Leica Aperio image analysis software. Pixels were classified as negative, weak, medium, and strong positive, counted, and divided by the total number of pixels.
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