Aperio imagescope

Statistical analyses were performed using SigmaPlot software (version 13.0). The microscopy photograph analysis was performed with an Aperio ScanScope CS (Aperio Technologies, Vista, CA, USA), and the software of Aperio ImageScope (version 10.0) used for calculating the number of interesting particles. Data are presented as the mean ± standard deviation. Analysis of variance followed by Student’s t test or one-way ANOVA comparison adjustment was used, and statistically significant differences were defined as p < 0.05.

Triplicate cores were made of 118 endometrial tumors at our institution from hysterectomy specimens, and tissue microarrays were created. Immunohistochemical analysis DRD2 (1:300, Santa Cruze Biotechnology Inc.) was performed on 4-μmol/L sections of formalin-fixed, paraffin-embedded tissues using standard methodologies in UNC-CH Translational Pathology Laboratory Core. Individual slides were scanned using the Aperio™ ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio™ ImageScope. Non-parametric tests and Cox regression analysis were used to correlate DRD2 expression with clinical outcomes.
The mouse endometrial tumor tissues were formalin-fixed and paraffin-embedded at the Animal Histopathology Core Facility at UNC-CH. Slides (5 μm) were first incubated with protein block solution (Dako) for 1 h and then with the primary antibodies for Ki-67 (1:400), phosphorylated-S6 (1:300), VEGF (1:800) and BCL-2 (1: 1200) for 2 h at room temperature. The slides were then washed and incubated with appropriate secondary antibodies at room temperature for 1 h. Further processing was carried out using ABC-Staining Kits (Vector Labs, Burlingame, CA) and hematoxylin. Immunohistochemistry slides were scanned by Motic (Houston, TX) and scored by ImagePro software (Vista, CA).

TMAs were stained for CXCR6, CXCL16, and ADAM10 using methods we have described previously [14 (link),21 (link)]. Virtual slides were created with an Aperio ImageScope (Aperio Technologies) to analyze the immunohistochemical staining. True-color digital images of each stained sample were viewed using Aperio ImageScope v.6.25 software. An algorithm for determining the intensity of membrane-specific staining was used to calculate, for each sample, the staining intensity and percent of the target label by digitally analyzing the color intensity. A color markup image for each slide was obtained based on the membrane staining intensity. The output was viewed as determinations of staining intensity ranging from 0–3 to correlate with conventional manual scoring methods (where 0 = negative and 3 = strong staining), and statistical analyses were performed using the mean values.

Virus attachment was studied by virus histochemistry^{10 (link)}. In brief, tissue slides were deparaffinised with xylene, hydrated in graded alcohols to distilled water, and blocked for endogenous peroxidase in 0.3% hydrogen peroxide. Each tissue section was incubated overnight at 4 °C with 50 hemagglutinin units of purified formalin fixed FITC-labelled IAV or 1 × PBS (Medicago AB, Uppsala, Sweden) as negative control. FITC-labelled viruses were detected by a peroxidase labelled α-FITC rabbit polyclonal antibody (#ab19492, Abcam). The signal was amplified by a tyramide signal amplification kit (PerkinElmer AB, Upplands Väsby, Sweden). Peroxidase signal was revealed with 3-amino-9-ethyl-carbazole (Sigma-Aldrich AB, Stockholm, Sweden). Tissues were counterstained with hematoxylin (Sigma-Aldrich), mounted with Vision Mount (Thermo Fisher Scientific) and scanned using Aperio Image Scope (Aperio Technologies, CA. U.S.A.) using a 20x objective. Two independent observers scored all digital virus histochemistry images. The percentage of stained cells of a given cell type in each tissue was scored according to a 3-tiered scale: “−” (<1% stained cells), “±” (1–50% stained cells), or “+” (>50% stained cells).

The specimens were scanned using an Aperio ScanScopeCS2 (Leica Biosystems/Leica Micrisystems KK) and were analyzed using the Aperio ImageScope software program (Aperio Technologies, Inc.). Basically, the percentages of all nucleated bone marrow cells (ANCs) that were positively stained for each antibody were counted at ×20 magnification to determine the immunohistological score of 0–100%.
c)RAEB: refractory anemia with excess blasts

The parathyroid glands were fixed with 10% (vol) neutral-buffered formalin and routinely processed with paraffin. The paraffin blocks were sectioned into slices of approximately 5 μm in thickness. After dewaxing and rehydration, the sections were incubated with 3% (vol) hydrogen peroxide/PBS, to quench the endogenous peroxidase, and treated with 0.05% (w/v) pronase E for deproteinization. The sections were incubated with an anti-BrdU antibody (Clone: Bu20a; DAKO Inc., Carpinteria, CA) and with horseradish peroxidase-labelled polymer conjugated to anti-mouse immunoglobulins (Envision+; DAKO Inc.). The signal was visualized with 3,3-diaminobenzidine. Hematoxylin was applied for nuclear counterstaining.
The ratio of BrdU-positive cells was calculated from the number of BrdU-positive nuclei, the number of total nuclei, and the area of a section. The area of parathyroid gland cells was expressed as the cell size and determined using a hematoxylin-stained section. The cell size was calculated using the following formula: Cell size (μm²) = the total area of section/the total number of nucleus in a section. These parameters were analyzed using the Aperio ImageScope and Nuclear (ver. 9) software programs (Aperio Technologies Inc., Vista, CA).