Tissuequest system

Paraffin-embedded tissue microarrays (TMAs) used to survey IL-8 expression is composed of 91 tumor resection specimens of pancreatic cancer that include 10 adjacent normal/cancer pairs. Adjacent normal and cancer sites in donor paraffin blocks were identified by an experienced pathologist using matching hematoxylin and eosin reference slides, and the TMAs were constructed in duplicate or quadruplicate cores, each 2 mm in diameter. TMA sections were stained with anti-human IL-8 antibody (1:500 dilution; Abcam, ab7747, Cambridge, UK) followed by incubation with the appropriate fluorescently conjugated secondary antibody (1:1000 dilution; Abcam, ab150080, Cambridge, UK). Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; Molecular Probes, D3571, Eugene, OR, USA). The full-field fluorescence image of each core was acquired using the FACS-like Tissue Cytometry system (Tissue Gnostics, Vienna, Austria). The percentage of positive cells in each image was further quantified by TissueQuest system and the software TissueFAX (Tissue Gnostics, Vienna, Austria). Tumor heterogeneity was evaluated using Pearson’s correlation coefficient (r) in two different cores from the same tumor blocks as described previously [16 (link)].

FFPE PC tissues or TMA blocks were cut into 5-μm-thick sections and stained by H&E or stained with anti-human CD8 antibody (1:100 dilution; M7103, DAKO, Carpinteria, CA, USA), anti-human PD-L1 antibody (1:50 dilution; ab205921, Abcam, Cambridge, MA, USA), anti-human PD-1 antibody (1:50 dilution; ab117420, Abcam), anti-human CD44 antibody (1:500 dilution; M7082, DAKO), or anti-human CD133 antibody (1:500 dilution; 3663S, Cell Signaling, Danvers, MA, USA) at 4 °C overnight. TMA sections were followed by incubation with appropriate fluorescently conjugated secondary antibodies for IF at room temperature for 1 hour and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. Slides for immunoreaction products of IHC were followed by incubation HRP-conjugated secondary antibodies, developed with DAB chromogen system (DAKO), and counterstained with hematoxylin. The full-field images of IHC-stained slides or IF-stained TMAs were acquired using the FACS-like Tissue Cytometry system (Tissue Gnostics, Vienna, Austria). The percentage of CD8-, PD-L1-, or CD44/CD133-positive cells in each fluorescence image was quantified and calculated the average of cell percentages in duplicate or quadruplicate cores per patient by TissueQuest system and the software TissueFAX (Tissue Gnostics, Vienna, Austria) as described previously [61 (link)].