Aperio scanscope xt slide scanner

The lungs and nasal turbinates of mice (n = 3) were collected at 3 and 5 dpi and fixed via intratracheal infusion and immersion in a 10% neutral-buffered formalin solution. Tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Immunohistochemical staining was performed on serial histologic sections to determine the distribution of AIV antigens. A goat primary polyclonal antibody against influenza A/USSR/1977 (H1N1) virus (US Biological, Swampscott, MA; diluted 1:1000) was used on tissue sections that were previously subjected to antigen retrieval for 30 min at 98 °C with Dako Target Retrieval Solution, pH 9 (Agilent, Santa Clara CA). To quantify the extent of viral infection in the lungs, digital images of whole-lung sections stained for viral antigens were acquired with an Aperio ScanScope XT Slide Scanner (Leica Biosystems, Buffalo Grove, IL). Both uninfected and virus-positive regions were then manually outlined, and the areas of the outlined regions were determined with ImageScope software (Leica Biosystems).

After the final scan, rats were placed under deep anaesthesia (2.5%-3% of isoflurane (Pharmachem, Eagle Farm, Australia) in 100% oxygen at the rate of 1L/min) and euthanized by intracardiac perfusion of 100 mL of phosphate buffered saline 1X (Sigma-Aldrich, Castle Hill, Australia) and then 100 mL of 10% buffered formalin solution (Thermo Fisher Scientific Australia Pty Ltd, Scoresby Vic, Australia). The brains were fixed for 24 hours in 10% buffered formalin solution and stored at room temperature in Ethanol (70% v/v, Sigma-Aldrich, Castle Hill, Australia).
The brains were embedded in paraffin and axial sections of five μm thickness were cut. The slides were stained using Luxol Fast Blue with Cresyl Violet on the Leica Auto—stainer XL (Leica Biosystems, Wetzlar, Germany) to visualize neuronal organization of the brain tissue and myelination in the white matter. High resolution images (40 × magnification) were obtained by scanning the glass microscopic slides using an Aperio Scan Scope XT Slide Scanner (Leica Biosystems, Wetzlar, Germany).

TMAs were cut in 4 micrometer thick sections and used for immunohistochemical staining, as previously described [16 (link)]. The immunohistochemically stained and mounted slides were scanned using an Aperio ScanScope XT Slide Scanner (Aperio Technologies, Vista, CA) for generation of high-resolution digital whole slide images, followed by annotation by certified pathologists. In brief, the manual score of IHC-based protein expression for all proteins screened was determined as the fraction of positive cells defined in different tissues: 0 = 0–1%, 1 = 2–25%, 2 = 26–75%, 3>75% and intensity of immunoreactivity: 0 = negative, 1 = weak, 2 = moderate and 3 = strong staining. All annotation and immunohistochemical data for the screening TMA together with validation data for of all primary antibodies is publically available in the Human Protein Atlas (www.proteinatlas.org) [28 (link)]. Primary antibodies used for immunostaining of validation TMAs included HPA055214 (dilution 1:250) for detection of Transmembrane protein 79 Tmem 79) and HPA035392 for detection of Acyl-CoA oxidase-like protein (ACOXL) (dilution 1:1000).

Digital images of the entire array of spots were captured using the Aperio ScanScope XT Slide Scanner (Aperio Technologies, Vista, CA, USA) under 20 × objective magnification. A quantitative scoring of PAR2 expression of the whole spot was analysed using the positive pixel algorithm of Aperio ImageScope [45 (link)]. Based on the staining intensity of the tissue, the Aperio Positive Pixel Count v9 algorithm generates an automated positive pixel count (positive and negative pixels %). The intensity output was exported to an Excel spread sheet and the difference in expression intensity (%) between normal and tumour regions were calculated. GraphPad Prism 6 (GraphPad Software) was used to calculate significance and generate graphs. Graphs were generated to show the % expression change for tumour versus normal kidney. Since there were only two groups (normal and RCC), significance of differences was analysed with Student’s t-test. P<0.05 was considered significant. For quality assurance, all TMA spots were further evaluated visually by a pathologist to provide a semi-quantitative estimate of staining intensity.

Joints were removed, fixed in 10% formalin for 24 hr, decalcified in 5% trichloroacetic acid for 7 days, dehydrated, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E; to determine inflammatory cell influx and chondrocyte death) or safranin O and Fast Green (S&F; to determine proteoglycan depletion and cartilage and bone destruction). Digital images of whole H&E- or S&F-stained slides were captured using the Aperio ScanScope® XT Slide Scanner (Aperio Technologies, Inc. Vista CA) system. Histopathological features of arthritis were examined by two blinded independent investigators. A histopathologic score assigned to paws was based upon the extent of inflammation, pannus formation, cartilage damage, and bone erosion, each using a scale of 0 (none to minimal) to 3 (the most severe) [29 (link), 30 (link)]. At least 4 fields/paw at 10x of the original scanning were scored and averaged. Paws were individually scored and total maximum score for each paw is 12. Two hind paws/mouse were analyzed. The mean arthritic histopathologic score for each experimental group was constructed by dividing the sum of the total score of each paw in the group by the number of paws in each experimental group.