The distribution pattern of cells expressing RANKL, OPG and ALP was also assessed in paraffin-embedded tibia sections. Briefly, after deparaffination, sections were rehydrated in graded ethanol and incubated in 4% bovine serum albumin (BSA) and 3% sheep serum to block unspecific immunobinding, based on Pérez-Baos et al.24 (link). Mouse monoclonal antibodies against RANKL, ALP (Santa Cruz Biotech, Santa Cruz, CA, USA) or OPG (R&D Systems, Minneapolis, MN, USA) (at 1:200 dilution) were added overnight, at 4 °C. This was followed by incubation with corresponding biotinylated goat anti-mouse IgG (GE Healthcare, Little Chalfont, Buckinghamshire, UK), at 1:200 dilution, and peroxidase ABC with 3,3′-diaminobenzidine tetra-hydrochloride as chromogen (Dako, Golstrup, Denmark). Sections were counterstained with hematoxylin, mounted in DPX medium (VWR International, Leuven, Belgium) and photographed using an automated iScan Coreo slide scanner (Ventana Medical Systems, Oro Valley, AR, USA) as previously described24 (link). Three random areas per slide were selected blinded to group assignment and quantified with Image J software24 (link). Results are expressed as a percentage of positive stained area in relation to the total tissue area. The negative controls involved incubation with an IgG isotype.
Iscan coreo
Manufactured by Roche
Sourced in United States
The IScan Coreo is a laboratory instrument designed for automated imaging and analysis of biological samples. It provides high-resolution digital imaging capabilities for a variety of applications, enabling users to capture, store, and analyze images of their samples.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark xt, by Roche (3 mentions)
Peroxidase abc with 3 3 diaminobenzidine tetra hydrochloride, by Agilent Technologies (1 mentions)
Dpx medium, by Avantor (1 mentions)
Dpx mounting medium, by Avantor (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Ncl l end, by Leica (1 mentions)
Image viewer, by Roche (1 mentions)
Dako pd l1 22c3 pharmdx kit, by Agilent Technologies (1 mentions)
Dako autostainer link 48 platform, by Agilent Technologies (1 mentions)
Ultraview detection kit, by Roche (1 mentions)
Clone 30 9, by Roche (1 mentions)
15 protocols using iscan coreo
1
Quantifying RANKL, OPG, and ALP Expression
2
Histological Analysis of TA Muscle
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Two consecutive 5 μm TA muscles sections were oriented in the same plane and attached to a single microscope slide (Superfrost PLUS; Fisher Scientific). H&E stained TA cross sections were photographed using an automated iScan Coreo slide scanner (Ventana Medical Systems, Tucson, AZ, USA) with 200× magnification. The cross-sectional diameter (CSD) and cross-sectional area (CSA) were measured using the Image J software package 1.49v (NIH, Bethesda, MD, USA). CSD was measured using the Ferret’s diameter, defined as the maximum diameter across the lesser aspect of the section. Parallel horizontal lines at the top and bottom of the image ensured consistent perpendicular measurements. CSA was calculated after tracing the outline of the cross section. All measurements were taken manually and blinded to each group.
ATPase staining was performed in TA cross sections as described [16 (link)]. Briefly, cross sections were incubated for 45 min in 0.1 M glycine/NaCl buffer pH 9.4 with 0.75 M CaCl2 and 5 mg ATP (Sigma-Aldrich). After being rinsed in distilled water, sections were incubated in 2% cobalt chloride and then immersed in dilute (1/10) ammonium chloride to develop type 1 (white) and type 2 (black) fibers. After dehydration, sections were mounted with DPX mounting medium (VWR International Ltd., Lutterworth, UK).
ATPase staining was performed in TA cross sections as described [16 (link)]. Briefly, cross sections were incubated for 45 min in 0.1 M glycine/NaCl buffer pH 9.4 with 0.75 M CaCl2 and 5 mg ATP (Sigma-Aldrich). After being rinsed in distilled water, sections were incubated in 2% cobalt chloride and then immersed in dilute (1/10) ammonium chloride to develop type 1 (white) and type 2 (black) fibers. After dehydration, sections were mounted with DPX mounting medium (VWR International Ltd., Lutterworth, UK).
3
Muscle Fiber Atrophy Evaluation Protocol
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In order to assess fibers atrophy, a minimum of 200 muscle fibers per biopsy have been evaluated, comparing minimum transverse diameter and cross-sectional area of type I and type II fibers for relative prevalence. A threshold diameter lower than 30 μm (minimum value of the normal range for women) characterized atrophic fibers [14 (link), 15 ].
To calculate muscle areas H&E slides were scanned at 20x magnification by Iscan Coreo (Ventana, Tucson, AZ, USA). Areas of skeletal muscle and connective and adipose tissue were identified by a pathologist for each muscle biopsy and analyzed by Image Viewing software (Ventana, Tucson, AZ, USA).
To calculate muscle areas H&E slides were scanned at 20x magnification by Iscan Coreo (Ventana, Tucson, AZ, USA). Areas of skeletal muscle and connective and adipose tissue were identified by a pathologist for each muscle biopsy and analyzed by Image Viewing software (Ventana, Tucson, AZ, USA).
4
Quantitative Analysis of Tumor Vasculature
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To characterize the tumor vasculature, the mean intratumoral MVD was quantitatively assessed according to immunohistochemical CD34 staining. We obtained formalin-fixed specimens embedded in paraffin of the 15 patients who underwent surgical tumor resection by thoracotomy. The specimens were cut into 4-μm slices and fixed to histology slides (X-tra, Leica Biosystems, Nussloch, Germany). After hematoxylin/eosin staining, the slides were stained with CD34 antibody (1:30, NCL-L END, Novocastra, Leica Biosystems, Nussloch, Germany) using an automated staining system (BenchMark XT, Ventana Medical Systems, Oro Valley, AZ, USA). The slides were scanned with a slide scanner (iScan Coreo, Ventana Medical Systems). A pathologist blinded to clinical and imaging data performed the histopathological analysis by visually counting the positive microvessels on the scanned images using public domain software (imageJ, http://rsbweb.nih.gov/ij ).
5
Standardized Ki67 Scoring Methodology
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A single experienced pathologist (D.C.A.) conducted the real-time Ki67 scoring for ACOSOG Z1031B. If the estimated rate was low (< 2.7%), or high (> 10%), a whole slide estimate was conducted. If the score was between 2.7% and 10%, point counting was conducted using an ocular grid, at least three high-power fields with a minimum of 100 cells scored. Retrospective analysis of ACOSOG Z1031A used the iScan Coreo scanner (Ventana) with the Companion Algorithm Ki-67 (30-9) software. The imaging approach required three to 10 areas of interest be selected at 4× magnification excluding ductal carcinoma in situ, vessels, and lymphocytes and avoiding perinecrotic or necrotic areas. The image analysis result was reviewed to ensure the software was correctly differentiating between benign and malignant cells, and, if not, the case was triaged to visual point counting. The visual point counting approach required color photomicrographs with a background grid taken at 40× of at least three fields selected based on invasive tumor content and the quality of the histology, not on Ki67 staining pattern, to obtain tumor cell count of at least 200. The scorer counted the total number of tumor cells and the number of Ki67-positive cells that intersected with first grid line and every third gridline thereafter.
6
Correlating Multimodal Imaging and Histopathology in Prostate Cancer
7
Quantitative Assessment of Tissue Fibrosis
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For histology, Masson’s trichrome was used. It stains the cytoplasm in pink, the nuclei in black and highlights the collagen fibers in green (Figure 1 A and Figure 2 A). It was used to locate and quantify the area of fibrosis after digital scanning of these slides (iScan Coreo, Roche Ventana, Meillan, France) (Figure 1 B and Figure 2 B). The quantification was based on DIA using the public domain National Institutes of Health (NIH) Image program ImageJ (available at http://rsb.info.nih.gov/nih-image/ ). In this process, the histological image was binarized to eliminate the background of pure paraffin. Then, a color deconvolution in three channels was performed allowing the identification of collagen. The ratio of the fibrosis area to the total area gave the percentage of fibrosis in the sample.
8
Digital Pathology Slide Scanning for Targeted Cancer Analysis
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A digital pathology slide scanner (iScan Coreo, Ventana Medical Systems, Inc.) was used for image-based documentation of the marked cancer region to be scraped and analyzed. First, the original H&E stained slide generated in step (a) was digitally scanned. The scanned image was magnified up to 20 times, the cancer area was marked, and calculated using image viewing software (Image Viewer, Ventana Medical Systems, Inc.). Furthermore, the first and second Gleason patterns were noted. Then the cancer area marks were copied from the image and marked back onto the original H&E stained glass slide using a microscope, serving as a ‘map’ that guided the cancer cells scraping procedure.
9
Pilot Study of Breast Pathology Diagnosis
10
Pilot Study of Breast Pathology Diagnosis
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A cohort of cases from the Breast Pathology Study (B-Path) was used for this analysis. The methods for test case identification and the development and recruitment of pathologists have been previously described (3 (link), 7 (link)). Briefly, single, representative diagnostic slides from excisional or core breast biopsies of 180 women were included in this pilot study. Each slide was digitally scanned (iScan Coreo, Ventana Medical Systems, Tucson, AZ), and a whole slide image (WSI) was created, allowing the digital virtual slide to be viewed, magnified, and annotated on a computer using a web-based viewer.
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