Aperio scanscope scanner

Sections of 4% paraformaldehyde-fixed paraffin-embedded tumor slides were stained with anti-human DR5 rabbit polyclonal antibody or rabbit IgG control. ABC kit (Vector Laboratories, Burlingame, CA, USA) and DAB kit (Vector Laboratories) were used to develop the signals from antibody staining. Slides were counterstained by hematoxylin (Vector laboratories). Glass slides were scanned with Aperio ScanScope scanner (Leica Microsystems Inc., Lincolnshire, IL, USA) and were analyzed with automated image analysis algorithm Aperio Positive Pixel Count (Leica Microsystems Inc.) using the default set of parameters. Briefly, in each slide, tumor cell areas were distinguished from neighboring stromal cells by the higher nucleus to cytoplasm ratio (N/C ratio) of cancer cells. In selected tumor areas, averaged intensity of staining was acquired through dividing total intensity by the sum of the numbers of weak positive (N_wp), positive (N_p) and strong positive pixels.

Stained slides were digitalized at 40× magnification using an Aperio ScanScope scanner (Leica Biosystems, Richmond, IL, USA). Expression levels of EBER and histological marker proteins were digitally assessed in four specified regions of interest (ROIs), which contained the laryngeal mucosal lesion and submucosal tissue, using semi-quantitative image analysis (Tissue Studio v2.1; Definiens AG, Münich, Germany) [7 (link),25 (link),33 (link)]. The image analysis software allowed for the specific mining of staining intensities in the cell nuclei (EBER and MYC) or whole cells (p16INK4a, BCL-2, B2M, CD3, and CD161); nuclei and cells were categorized as “negative” or “positive” (weak, moderate, or high staining intensity). The positivity index (PI; %) ((number of positively stained cells or nuclei/total number of cells or nuclei) × 100) was calculated. The same investigator who was blinded to the clinical outcomes chose the ROIs and performed a computer-supported evaluation.

As mentioned above, IHC was used to measure the expressions of E-cadherin (CAT # E-CAD-L-CE; Leica Biosystems Ltd., Newcastle, UK) and CD1d (CAT # orb11652; Biorbyt, Cambridge, UK). Sections of tissue microarrays were automatically stained. For optimal detection, the antibodies were diluted at 1:100. Antibody reactions were performed at room temperature for 30 min (E-cadherin) and 20 min (CD1d) [37 (link)]. Using an Aperio ScanScope scanner (Leica Biosystems, Richmond, IL, USA), the stained slides were digitized at 40× magnification. The presence of H. pylori and the cellular expression levels of E-cadherin and CD1d were digitally assessed in four specified regions of interest via semi-quantitative image analysis (Tissue Studio v2.1; Definiens AG, Münich, Germany) [44 (link)]; the selected regions contained the laryngeal mucosal lesion and submucosal tissue, and passed the filtering process because of a cell count ≥ 15 [44 (link),45 (link)]. A histological score (1 × percentage of cells positive for low brown chromogen intensity) + (2 × percentage of cells positive for medium brown chromogen intensity) + (3 × percentage of cells positive for high brown chromogen intensity) [46 (link)] was calculated for cellular staining by the scientific team members (T.-C.C. and C.-G.H.) without knowing the clinical information [36 (link),37 (link)].

Once fixed, the in vivo constructs were dehydrated in successive baths of increasing concentrations of ethanol and embedded in paraffin as previously described with an automaton (Laboratory of Pathological Anatomy, CHU)^{30 (link)}. Then, 4 µm sections were prepared using a microtome and mounted on silanized slides for immunostaining. The sections were deparaffinized by successive baths of toluene (two baths of 5 min), 100% ethanol (two baths of 5 min), 90% ethanol (5 min), 70% ethanol (5 min) and finally distilled water. The deparaffinized sections were stained with hematoxylin-eosin-safran (HES) and alizarin red S (2%, pH 4.1) according to routine protocols. Samples were mounted with Eukitt (DAKO). An Aperio ScanScope scanner (Leica Biosystems) was used to digitalize the histological slides.

Liver sections were fixed in 10% neutral buffered formalin before standard histological processing, sectioning, and staining with hematoxylin and eosin (Richard-Allan Scientific). Several sections of the evaluated tissues were placed together on the same slide to provide representative regions of the entire organ for the histopathology analyses. Slides were then evaluated with a Nikon Eclipse Ni microscope and then digitized to scalable images up to a 20x objective lens with an Aperio ScanScope scanner (Leica Biosystems). Normal tissue, inflammation and clear space/glass were segmented, and the percentage of tissue area infiltrated by immune cells was quantified as well as the number of inflammatory foci on 2x magnification static images of the tissues using the FIJI image analysis program. Histological analysis was performed in a blinded fashion.