Aperio

PD-L1 expression was quantified by two observers jointly (DL &FF) evaluating the percentage of neoplastic cells with membranous expression at x200 magnification. The percentage of neoplastic stained cells was evaluated on the whole slide. We also assessed PD-L1 expression in immune cells and the localization of immune cells, within the neoplastic epithelium or within the submucosa below the dysplastic epithelium. CD8 expressing cells were characterized by their localization: within the dysplastic epithelium, in the sub-mucosal tissue, in both or absent. The percentage of CD8 positive lymphocytes were evaluated. The density of CD8 positive cells in the submucosa and in the epithelium were also evaluated on scanned slides (Aperio, Leica Biosystems, Vista, CA, USA) with QuPath Software (version 0.2.1, University of Edinburgh, Edinbugh, UK, available at https://qupath.github.io).

Collagen type-I from Achilles tendon of a 25 kg Yorkshire pig was studied. Pig tendon was chosen because it is a well-ordered structure and due to its size, it can be easily cut at various angles. The tendon was harvested from a healthy dead pig euthanized after an unrelated study with approval of the Animal Care Committee of the University Health Network, Toronto, Canada. The tendon was formalin fixed and cut along the tendon axis (longitudinal cut), at α = 30° (oblique cut) and perpendicular to the tendon axis (transverse cut). The samples were embedded in paraffin and cut into 5-μm thick sections. The sections were mounted on glass slides and stained with hematoxylin and eosin (H&E) to provide anatomical reference recorded with bright-field whole-slide scanner (Aperio: Leica Biosystems). One slide per cut angle was studied.

Immunostained slides for WT1 and LANA were scanned at 20x (Aperio, Leica Biosystems). Using HALO Imaging analysis software, WT1 and LANA staining were quantified as percent positive cells in areas involved by KS or adjacent uninvolved skin. Brown melanin staining along the epidermis was controlled for, as it was purposely excluded for WT1 analyses. WT1 was characterized based on percentage of positively stained cells, 1–20% (1+), 21–50% (2+), >50% (3+). Correlation analysis was assessed for proportions of five independent areas with high LANA or high CD8 cell numbers and the corresponding adjacent sections for other markers (LANA, CD8, CD4 and WT1). The number of cells and percentage of LANA+, CD4+ and CD8+ cells were quantified using HALO software with analysis tools, “Image Registration” and “Synchronization”. The total number of WT1+ cells, CD4+T cells, and CD8+T cells were quantified in LANA-rich areas. Conversely, five high CD8+ regions were selected and the total number of LANA+, CD8+, CD4+, and WT1+ cells were quantified in sequential sections. Proportional values of all indices were evaluated in both high LANA+ sections and high CD8+ T cells on corresponding sections with analysis performed as previously described [65 (link)]. These percentages were calculated and plotted using Graph Pad Prism version 9.4.1 for Windows (GraphPad Software, San Diego, CA, USA).

Histological studies were performed by standard protocols. Briefly, mice were sacrificed and their hearts were perfused with 20% KCl, fixed with zinc fixative solution (BD Pharmingen), and dehydrated with 30% sucrose. After embedding in paraffin, the samples were sectioned and processed for Masson’s trichrome staining. Images were captured with an Aperio (Leica Biosystems, Buffalo Grove, IL, USA) and infarct size was measured using Image J software.