Aperio cs2 scanscope slide scanner

Well-characterized anti-PD-L1 (clone 28–8; Abcam, Cambridge, MA, USA) and anti-cluster of differentiation 68 (CD68) (clone KP1; DAKO, Glostrup, Denmark) antibodies were selected. IHC was performed using an automated staining system (Leica Bond III; Leica Microsystems). The antibody dilutions were optimized to 1:100 for anti-PD-L1 and 1:400 for anti-CD68. The slides were dewaxed and rehydrated using distilled water, and were subsequently processed for PD-L1 (heat-induced antigen retrieval at pH 9.0) or CD68 (proteolytic treatment). After incubation with the primary antibodies (anti-PD-L1, 30 minutes; anti-CD68, 15 minutes), the tissue sections were rinsed, and the sections for PD-L1 staining were further incubated with EnVision FLEX+ Rabbit LINKER (DAKO) and EnVision+ HRP Labelled Polymer (DAKO). The sections for CD68 staining were incubated with the Bond Polymer Refine Detection Kit (Leica Microsystems). Staining was visualized using diaminobenzidine, and counterstaining was performed using hematoxylin. Formalin-fixed, paraffin-embedded tissue blocks of human placenta and tonsil were prepared as positive controls. The stained slides were scanned as whole-slide images using a ScanScope^® Aperio CS2 slide scanner (Leica Microsystems).

Spiral Arrays were constructed as described previously (Fig 1) [17 (link)]. Briefly, single blocks of tissue with the most representative tumor histology and of sufficient quantity was selected from each case. The corresponding H&E-stained slide was digitally scanned using a ScanScope^® Aperio CS2 slide scanner (Leica Microsystems, Melbourne, Australia). The scanned whole-slide image of each H&E-stained slide was reviewed to select and mark two continuous straight regions of interest (X and Y axes) prior to constructing the Spiral Arrays. Two 120.0-μm-thick sections were subsequently cut from each block and arranged together on the Spiral Array Constructor (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) as the X or Y axis on each section was aligned in the same direction. The aligned sections were rolled together into a cylindrical form and cut along the line reflecting the X and Y axes. One of the separated reels was embedded vertically into a recipient block to construct the Spiral Arrays. Finally, 4.0-μm-thick sections were prepared from the Spiral Array blocks for further histopathological evaluation using H&E staining and IHC.

For all histological analyses, formalin-fixed and paraffin-embedded samples were used. For fetal and adult liver analyses, the livers were sectioned transversal at 5 µm and mounted on Superfrost Plus Slides (R. Langenbrinck, Emmendingen, Germany). The stained slides were scanned with the Aperio CS2 ScanScope slide scanner (Leica, Wetzlar, Germany) at 20× or 40× magnification (Westdeutsche Biobank, University Hospital Essen, Essen, Germany) and images were exported as TIFF via Image Scope (Version 12.3.2.8013; Leica). Exported slides were opened (plugin “bioformats_package.jar.”) and analyzed using Fiji/ImageJ [53 (link)]. For the quantitative image analysis of special stains (Perls and PAS), the original exported Red–Green–Blue (RGB) image was split into an 8-bit image containing exclusively the information of one color of each staining: Perls Prussian Blue (blue) or Periodic Acid Schiff (magenta). Afterwards, each image was thresholded to find a binary black and white image, showing in white the staining of interest and in black the background. By using the “watershed” algorithm single iron particles (recognized by Perls) were separated, and the number and size were calculated with the “analyze particle” command. To calculate the glycogen containing area (recognized by PAS), the percentage of stained (white area) compared to unstained background (black area) was calculated.