Imagescope software

All tumor xenograft assays were performed using Rag2^−/−:IL-2Rγc^−/− mice (BALB/c background) as previously described [11 (link)],[22 (link)]. Animals were housed in a specific pathogen-free facility (Queen’s University Animal Care Services), with ventilated cages and sterilized food and water supply. All procedures with mice were approved by the Queen’s University Animal Care Committee. MTLn3 control, sh-p53 and sh-p53/sh-Toca-1 cells were grown to 70 to 85% confluence before trypsinization and counting. For xenograft assays, 0.5 × 10⁶ cells were injected into the right thoracic mammary fat pads in a volume of 50 μl of 50% Matrigel using a hypodermic syringe. At end points of 4 weeks mice were sacrificed and primary tumor mass recorded. Several tissues were removed for detection of metastases, which were primarily observed in the lungs. The primary tumors and lungs from each mouse were used for histological analysis. Samples were fixed in formalin and embedded in paraffin, and 5 μm sections were stained with hematoxylin and eosin (H&E). H&E-stained sections were scanned using the Aperio CS digital slide scanner (Queen’s Laboratory for Molecular Pathology) and analyzed with ImageScope software (Aperio Technologies, Vista, CA, USA). The number of metastases and overall lung area were measured in a blinded fashion.

FFPE tissues of cases of GC with intestinal histology were used to construct tissue microarrays (TMAs) as previously described16 (link). Each samples was represented by three cores of 1 mm of diameter. TMAs were immunostained using the antibodies listed in Supplementary Table S1. Detection was carried out using Dako EnVision Plus-HRP kit (Dako). Slides were scanned with ScanScope® GL System (Aperio Technologies, Vista, CA) and visualized using ImageScope™ Software (Aperio Technologies). Immunoreactivity evaluation for each antigen is detailed in the Appendix (Methods section). FISH was carried out using HER2, MET and TOPO2A locus-specific and chromosome 17 centromeric probes (Vysis, Downers Grove, IL) diluted 1:100 in tDenHyb1 buffer (Insitus, Albuquerque, MN). The slides were examined using an Olympus BX61 (Bremerhaven, Germany) and the appropriate filters. Annotation of tumors was carried out as previously described17 (link)18 (link).

All slides were scanned using the ScanScope CS (Aperio Technologies, Inc., Vista, CA) and ImageScope software (Aperio Technologies, Inc.) was used to perform quantitative analysis. Quantitative analysis of lesion volume was performed on 8 sections taken from 5 slides per mouse, allowing for an analysis that represented of the whole brain. Quantification of Perls' positive reaction was performed similarly to the method used to calculate lesion volume; however, only the number of positive counts around the lesion was measured. The ImageScope Positive Pixel Count algorithm was used for quantification after the appropriate brain regions were outlined. To perform quantitative analysis of cortical Iba1 and GFAP immunoreactivity, four to five sections from five slides per mouse were selected. Microgliosis and astrogliosis were analyzed by placing identically sized boxes of 1000 × 1000 pixels in both the ipsilateral and contralateral motor cortex. Data is presented as the relative ipsilateral to contralateral signal for signal normalization per area quantified. For each section within the cortex, an area immediately lateral to the lesion was selected for quantification and the intensity of Iba1 and GFAP immunoreactivity was evaluated by means of relative positive pixel counts. An analyst blinded to the experimental groups performed the entire quantifying procedure.

Immunostained slides were scanned using the ScanScope XT system (Aperio Technologies, Vista, CA). Digitized images were captured using the ImageScope software (version 10.2.2.2319, Aperio Technologies) at 20× magnification.

HCC tissue samples with the corresponding adjacent non-tumor tissues were obtained from Qidong Liver Cancer Institute (Jiangsu province, China). Fresh specimens were immediately frozen and stored in liquid nitrogen for analysis after surgical resection from patients. Ethics permits were approved by the ethics committee of the local institute and informed consents were obtained from the patients involved in the study.
The commercially used TMAs containing a total of 80 pairs of HCC specimens with clinicopathological details were purchased from Zhuoli Biotechnology Co., Ltd (LVC1605, Shanghai, China). 76 pairs of HCC tissues were successfully stained. The clinicopathological characteristics of patients are available in Table 2. The relative intensity of positively expressed pNF90-S382 in both tumor tissues and non-tumor tissues was assessed by ImageScope software (Aperio Technologies, Vista, CA).

Formalin fixed cervical spinal cords were paraffin-embedded, cut 5 μm thick, and mounted on microscope slides. Sections were stained with hematoxylin and eosin (H&E; Richard-Allan Scientific, Thermo Fisher Scientific) and scanned into Imagescope software (Aperio Technologies, Inc., Vista, CA) using an Aperio Scanscope® CS2 instrument (Aperio Technologies, Inc.). For each section, 3 images of randomly selected fields of white matter were taken at × 40 magnification. Each image was then uploaded onto Fiji/Image J software (National Institutes of Health, Bethesda, Maryland), and cells were counted according to the number of pixels and circularity. For each mouse section, 3 fields were measured for cellular infiltrates per group (n = 5).
Luxol fast blue (LFB; IHC World, LLC, Woodstock, MD) staining of spinal cord sections was analyzed using pixel-based algorithms by Imagescope software. For each group, 2 sections for each mouse and 5 mice per group were analyzed to quantify the levels of myelin. Myelin levels were calculated as the percent positive pixels/total number of pixels × 100, and al histological measurements were represented as the mean ± SEM.

Cresyl violet staining was performed to measure infarct volume at 28d post-tMCAO, as described in our previous work [29 (link)]. Mice were euthanized at 28d post-stroke and transcardially perfused with 10 mL of saline containing 5 mM EDTA using a peristaltic pump at a speed of 5 mL/min, followed by 30 mL of 4% PFA in PBS. Brains were collected and post-fixed in 4% PFA for 24 h, then transferred to PBS for storage at 4 °C. Brains were cut coronally into a series of 30-μm-thick sections on a semi-automated vibrating microtome (Compresstome^® Model No. VF310-0Z, Precisionary Instruments; Natick, MA, USA). Cortical infarct volume was quantified from infarcted areas in ten brain sections spaced 0.5 mm apart using Cresyl violet staining. Sections were scanned using an Aperio ScanScope^® CS system and analyzed with ImageScope Software (Aperio Technologies; Vista, CA, USA). The border between infarcted (dark purple stain) and non-infarcted area (light purple stain) was outlined and quantified using the software. Infarct volume was calculated by subtracting the area of healthy tissue on the ipsilateral hemisphere from the total area of the contralateral hemisphere.